Effects of neuraminidase on the phenotype of sindbis virus grown in fibroblasts obtained from patients with I-cell disease

Effects

SONDRA

of Neuraminidase on the Phenotype Fibroblasts Obtained from Patients SCHLESINGER,*

WILLIAM

S. SLYt

of Sindbis with I-Cell AND

IRENE

Virus Grown Disease

in

T. SCHIJLZE$

Sindhis virus grown in fihrohlasts obtainerI from patients with I-cell disease shows an cxaggrratetl sensitivity to freezing and thawing. Vesicular stomatitis virus, hut not influenza virus, has the same phenotype. Treatment of I-crll Sindhis virus with neuraminidase proteds t hc virus f’ronr inactivation and is ahlr to reactivate virus previously inactivated t)y frtwing anti thawing. I-cell fihroblasts haw heen shown to contain iwrrased levels of twund sialic, acid. Although the Lccll viral gl,vcoprotcins cont;kwd sialic, ad, thcw wx+ no c5Gience for ~xwss sialic acid in the glvqwptides isolated from I-cell Sindhis viral fil~cwprotcins. l’ronaae digestion of l-cell viral glycoproteins released four glycopeptitlc~s IS ,-S,) which co-chromatogral)hed on Bio-Gel 1% with those released from glycoproteins of control .Sindhis virus. In hot h cases, trcatmc>nt of the glycopcptitles with ncwraminiclase ~~illlhd tllhappC”Ll~an~r of s, and s ,( condent with the removal of 2 and I residues of sialic ac%l from t hcsc glycopeptities. A second phenot,vpic abnormality ot 1.~~11 Sindbis ~irrls-itlc,renst,~l sensitivity to ‘I’riton X-IOO-was not affected t)y trrwtment with neur~minidase Thus. whereas the frwzts-t haw sensitivity of l-cell Sindhis virus may he rc~latetl to an increase in sialic acid containing ~lycoli~kls, the ‘I’riton X- 100 sensitivity may be asswiatctl with other phenotypic alterations in the viral mrmhranr. Sindhis virus cbtainrtl from fihrohlasts from a patient with mucolipidosis I. a disease ;wsociatrd with an isolated rl~fi&~ncv in nruraminidasc~. is miltllv freeze-thaw wnsitiw. hut dors not show an increased icsnsitivitv to l’riton .XIOO.

l-cell disease (mucolipidosis II) is a severe inherited lysosomal storage disease characterized clinically by profound psychomotor retardation, severe Hurler-like skeletal changes but without mucopolysacchariduria, progressive limitation of
amounts of these hydrolases into the culture medium (Wiesmann and Herschkowitz, 1974). These enz:ymes were found to be defective in recognition and uptake by fibroblasts (Hickman and Neufeld, 1972), and a defect in processing a carbohydrate recognit,ion marker common to many lysosomal enzymes was postulated (Hickman and Neufeld, 1972; Hickman et rd., 1974). Recent evidence suggests that t,his common recognition marker on normal lvsosomal enzymes is a phosphomannose mo>ety (Kaplan et al., 1977). Neuraminidase has recently been added to the list of lysosomal enzymes for which these cells are deficient and a number of previous observations on I-cell disease may be explained by this defect (Thomas et cd., 1976; Cantz et al., 1977). These observations include reports of increased levels of, and decreased ability to metabolize, the

410

SCHLESINGER.

SLY,

sialic acid-containing glycosphingolipids, Go:3 and GM:,, in I-cell fibroblasts (Dawson et al., 1972), electrophoretic abnormalities of I-cell fibroblast enzymes (Vladutiu and Rattazzi, 1975), large increases in sialic acid-rich oligosaccharides in urine from Icell diseasepatients (Michalski et al., 1977), and greatly increased levels of bound sialic acid in I-cell fibroblast extracts (Thomas et al., 1976; Cantz et al., 1977). The relationship of the neuraminidase deficiency and the deficiency of the other lysosomal enzymes in I-cell libroblasts to t.he basic biochemical defect in the disorder is still unclear. We suspected a membrane abnormality in I-cell fibroblasts on the basis of their unusual freeze sensitivity and tested this possibility by comparing the properties of Sindbis virus grown in normal and I-cell libroblasts (Sly et al., 1976). Sindbis virus is an enveloped RNA virus that matures by budding through the host plasma membrane from which it derives the lipid components of the viral envelope (Pefferkorn and Shapiro, 1974). The envelope has only two proteins, both glycoproteins. The amino acid sequence of these proteins is specified by the viral genome, but the carbohydrate components are determined by the host glycosylation enzymes. Our earlier report showed that Sindbis virus grown in I-cell libroblasts was phenotypically altered, as evidenced by greatly exaggerated sensitivity to inactivation of the virus by freezing and thawing, and by treatment with the nonionic detergent Triton X-100. We proposed that the abnormal properties of I-cell Sindbis virus were due to the incorporation of altered host cell components into the viral envelope during the assembly of the virus. The results reported here extend the original observations. We have found that vesicular stomatitis virus (VSV), but not influenza virus, showed the same phenotypic changes as Sindbis virus after growth in I-cell fibroblasts. Furthermore, Icell Sindbis virus could be “cured” of its sensitivity to freezing and thawing by treatment with neuraminidase. MATERIALS

AN13

METHODS

Cells. The human fibroblasts, listed as S291, S235, and S186, represent strains ob-

AND

SCHULZE

tained from human adults that were normal with respect to I-cell disease. L.T. and T. M. are designations of fibroblasts obtained from two patients with I-cell disease. The human fibroblasts with neuraminidase deficiency are those described by Kelly and Graetz (1977). They were kindly provided to us by Dr. Kelly. The conditions for growth have been described previously (Sly et al., 1976). Virus. Sindbis virus is the variant isolated by its ability to grow in mouse myeloma cells (Symington and Schlesinger, 1975). Stocks of the virus were prepared on mouse L cells because the latter cells were more readily available to us. Vesicular stomatitis virus was obtained from Dr. A. Huang and was passaged on chicken embryo fibroblasts. The protocols for infection of human fibroblasts with Sindbis virus and VSV were identical to those described previously (Sly et al., 1976). The influenza virus used was the WSN strain of influenza A(HON1). Stocks were grown in MDBK cells (Madin and Darby, 1958) as described by Choppin (1969). Human fibroblasts were infected by adding the virus to washed monolayers at a multiplicity of 0.5 per cell. After 1 hr at 37”, the inoculum was removed and MEM with 10% fetal calf serum was added. Moderate cytopathic effects were observed after approximately 24 hr at 37” and maximum virus yield of approximately 10” PFU/ml were obtained after 30 hr. Virus suspensionswere freed of cellular debris and used as described in Results. Infectivity assays were carried out on chick embryo fibroblasts as previously described (Noronha-Blob and Schulze, 1976). Enzymes

and

chemicals.

Vibrio

cholera

neuraminidase was obtained from Behring Diagnostics, Somerville, N. J. Escherichia coli alkaline phosphatase was a gift from Dr. M. J. Schlesinger. 2-Deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid was the material provided by Dr. Peter Palese (Meindl et al., 1974) and was given to us by Dr. Eve Briles (Washington University). Triton X-100 was purchased from Sigma Chemical Company, St. Louis, MO. Purification of Sindbis virus. Treatment of Sindbis virus with neuraminidase was always carried out on gradient-purified vi-

NEURAMINIDASE

AND

rus. The procedures for labeling the virus with [““Slmethionine have been described (Sly et al., 1976). The virus was purified either by centrifugation to equilibrium in a sucrose gradient (Sly et al., 1976) or by rate-zonal sedimentation as in Fig. 1. Freezing and thawing of virus. Freezing of virus was carried out by placing well sealed glass tubes containing the virus in a dry ice-acetone bath. The samples were thawed by placing them at 37”. Analysis of Sindbis glycopeptides. The preparation of [‘“Cl or [“Hlglucosamine-labeled Sindbis virus was described previously (Sly et al., 1976). Pronase digestion, neuraminidase treatment, and gel filtration followed the methods of Sefton and Keegstra (1974) and Keegstra et al. (1975). RESULTS

Properties of VSV and influenza virus obtained from I-cell fibroblasts. We had previously reported that Sindbis virus grown in I-cell fibroblasts is extremely sensitive to freezing and thawing (Sly et al., 1976). The virus titer dropped over loo-fold after three cycles of freezing and thawing. In order to determine if this observation could be extended to other viruses, we grew

I./

I

I1

3

5

7

I

9

I1

II 13 15 17 TUBE NUMBER

2,

I9 21

23

25

27

FIG. 1. Rate zonal sedimentation of Sindbis virus. The medium from either I-cell or S186 fibroblasts infected for 16 hr with Sindbis virus was harvested and divided into two fractions. One part was frozen and thawed three times (Table 3). Each sample was then subjected to centrifugation in a 15 to 30% sucrose gradient for 2 hr at 25,000 rpm in a Beckman model 1,3-50 ultracentrifuge. Tube 1 is the bottom of the gradient. W control, l - - -0 frozen and thawed.

I-CELL

SINDBIS

VIRUS

411

VSV and influenza virus in I-cell and normal human fibroblasts and tested the stability of these viruses to freezing and thawing. The data presented in Table 1A show that I-cell VSV behaves like Sindbis virus: the I-cell virus is inactivated by freezing and thawing, whereas the virus from normal fibroblasts is hardly affected by this treatment. In contrast, I-cell influenza virus is not inactivated by freezing and thawing (Table 1B). Effect of neuraminidase on inactivation of Sindbis virus by freezing and thawing. One well recognized distinction between influenza virus and viruses such as Sindbis virus and VSV is the absence of sialic acid on the former. Based on this difference, we examined the effects of neuraminidase on I-cell Sindbis virus. We have used Sindbis virus because we obtained better yields with this virus grown on human fibroblasts than with VSV. Treatment of Sindbis virus with neuraminidase followed by freezing and thawing resulted in essentially complete protection of the virus from inactivation (Table 2). These experiments were performed with fibroblasts obtained from two different patients (L.T. and T.M.) with Icell disease. In both cases, Sindbis virus was inactivated by freezing and thawing and was protected from inactivation by prior treatment with neuraminidase. In order to show that protection was actually due to neuraminidase activity, we carried out the incubation with neuraminidase in the presence of the neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA) (Meindl et al., 1974). In the presence of 1 mM

412

SCHLESINGER,

SLY,

FANA, neuraminidase was unable to protect the virus from inactivation by freezing and thawing. We also tested another hydrolytic enphosphatase. Incubation zyme, alkaline with this enzyme did not stabilize the virus against inactivation by freezing and thawing (Table 2B). Treatment with neuraminidase not only protected I-cell Sindbis virus from inactivation by freezing and thawing but also reactivated the virus after inactivation. In order to demonstrate reactivation, the culture medium containing Sindbis virus harvested from either I-cell or normal human fibroblasts was divided into two fractions; one-half of each was subjected to three cycles of freezing and thawing (Table 3). The samples were then centrifuged in a sucrose gradient (Fig. 1). A portion from the peak of radioactivity for each of the samples was titered for infectivity. Three of the samples were incubated at 37” for 1 hr in the presence or absence of 50 units of neuraminidase and then titered again. Treatment of the I-cell virus with neuraminidase led to a dramatic increase in titer (Table 3). Treatment of I-cell Sindbis virus with neuraminidase without freezing and thawing or treatment of normal Sindbis

AND

virus with the enzyme had very little effect on infectivity. We had considered that one possible explanation for inactivation of the I-cell virus was that freezing and thawing caused the virus to aggregate thereby reducing the titer. The finding that all of the virus samples sedimented at essentially the same rate before and after freezing and thawing demonstrated that this treatment does not lead to aggregation of I-cell virus. Effect of neuraminidase on Triton X-100 sensitivity of Sindbis uirus. I-cell Sindbis virus differed from the virus obtained from normal human fibroblasts not only in sensitivity to freezing and thawing, but also in inactivation by Triton X-100 (Sly et al., 1976). The latter property is retained after the virus is treated with neuraminidase (Fig. 2). We have examined the effect of neuraminidase on the Triton X-100 sensitivity of Sindbis virus obtained from fibroblasts from the two different I-cell patients and in both cases were unable to show any substantial difference in the degree of inactivation with or without neuraminidase treatment. These data suggest that the detergent sensitivity is not related to the presence of sialic acid on the virus. However, whatever the phenotypic change is that

TABLE THE

EFFECT

Source

OF NEURAMINIDASE

ON THE INACTIVATION THAWING”

of fibroblasts

Conditions

SCHULZE

2 OF I-CELL

of incubation

S~NDBIS ~.

VIRUS

BY FREEZING

..--

Neuraminidase (units added)

Titer of Sindbis virus (PFLJ/ml X 10’) Before”

A. I-cell

B. I-cell

C. I-cell

disease,

disease,

disease,

patient

patient

patient

L.T.

T.M.

T.M. .~-

None 1 hr, 37” I hr. 37” I hr 17” 1 hr: 37” 1 hr, 37” 1 hr, 37” 1 hr, 37” + 50 pg E. coli alkaline phosphatase 1 hr, 37’ 1 hr, 37” 1 hr, 37” + 1 m44 FANA

ANI)

After”

32 50

22 16

12.5 1.25

35 1.5

1%

0.125

12.5

12.5

..-

0.5 1 IO 0.02

12 0.3

5

0.003

9

0.08

16

0.4

19

9 0.2

3.3

’ Sindbis virus grown in I-cell fibroblasts was purifktl 1)y vcantrifugation to equilibrium in a sucrose gradient. This procedure and t hc conditions for freezing and thawing have brrn dcwribrd prwiorrsly. Incubation with neuraminidaw was carried out in 0.01 M Trih-Cl buffer, pH 7.0. The virus wiis titt~rrd on chicken embryo fibroblasts. ‘These titers of Sindbis virus are thp valuw obtained hefore or af’tcr three cwlrs of f’rcwing and thawing.

NEIJBAMINIDASE

AND

I-CELL

TABLE INACTIVATION

VIRUS

OF SINDBIS

BY FREEZING

SINDBIS

413

VIKlJS

3

AND THAWING TREATMENT

AND

REACTIVATION

BY NWRAMINIDASE

I-cell virus” 8.7 x 10” Control 4.3 i

Freeze-thaw 7.9 x 10:

lo9

Velocity gradient

Velocity gradient 3.1 x 10”

8.7 x 10”

I

2.6 x 10’

I

+ Neuraminidase

3.3 x 10”

S186 virus” 7.6 x 10’ Control

Freeze-thaw

3.6 x 10”

5.1 x 10”

Velocity gradient

Velocity gradient

1.2 x IO9

5 x 10” -

A+ Neuraminidase I 3 x lon ” The medium containing was then sedimented by rate sample was incubated at 37” the sample and are corrected

Neuraminidase I 3.5 x IOn

virus was divided in two. One-half was frozen and thawed three times. Each half zonal centrifugation in a sucrose gradient (Fig. 1). The radioactive peak from each with or without 50 units of neuraminidase. The numbers represent total PFIJ in for all dilutions.

confers sensitivity to Triton X-100 in Sindbis virus it does not affect influenza virus. Influenza virus obtained from I-cell and normal human fibrobiasts showed the same sensitivity to Triton X-100 (Fig. 2).

al., 1977). We have grown Sindbis virus in fibroblasts obtained from a patient with this disease.The virus was only moderately sensitive to freezing and thawing. The extent of inactivation as a function of the Properties of Sindbis virus obtained times the sample was frozen and thawed from fibroblasts with an isolated neurawas very similar to the results we have minidase deficiency. Mucolipidosis I is a otained previously with virus grown in filysosomal storage disease recently associ- broblasts from patients with mucolipidosis ated with a deficiency in neuraminidase, III (Sly et al., 1976). The virus obtained but without other lysosomal enzyme abnor- from neuraminidase deficient fibroblasts malities (Kelly and Graetz, 1977; Cantz et did not show an enhanced sensitivity to

414

SCHLESINGER,

SLY.

Triton X-100. Comparison of glycopeptides of Sindbis virus grown in I-cell or normal human fibroblasts. In our previous publication, we compared the glycopeptides obtained from normal and I-cell virus. These glycopeptides separate into four fractions (Sl, S2, S3, and S4) on a Bio-Gel P6 column (Sefton and Keegstra, 1974). We did not see any T r

jl

hd"

'8':

B Sindbir

AND

SCHULZE

difference in the ratio of the four glycopeptides from I-cell and normal Sindbis virus (Fig. 3). If the glycopeptides from the I cell virus had contained excess sialic acid, we would have expected them to be eluted earlier than the glycopeptides from the normal virus. The studies by Keegstra et al. (1975) with Sindbis virus obtained from chicken embryo fibroblasts or from baby hamster kidney cells showed that Sl and S2 differed from S3 only by the amount of sialic acid. Sl was reported to have 2 residues of sialic acid and S2 to have 1 residue. Treatment of the glycopeptides with neuraminidase converted Sl and S2 to S3. The Sl and S2 fractions of the glycopeptides of Sindbis virus obtained from both normal and I-cell fibroblasts show the same behavior; incubation with neuraminidase converts them to S3 (Fig. 3). These results indicate that the glycoproteins of Sindbis virus from I-cell and normal fibroblasts are similar with respect to sialic acid residues and suggest t,hat, if there is an alteration in the amount of sialic acid in I-cell Sindbis virus, it is in the amount of sialic acid in the glycolipid fraction.

“,,vr

DISCUSSION 001

002 % TRITON

X-100

FIG. 2. Treatment of Sindbis virus and influenza virus with Triton X-100. Sindbis virus was purified by centrifugation to equilibrium in a sucrose gradient. Aliquots of the virus were treated with neuraminidase (50 units per ml) for 1 hr at 37’. Neuraminidasetreated and control Sindbis virus were then incubated with the concentrations of Triton X-100 indicated on the graph for 15 min at room temperature and titered immediately afterward. In each experiment, a sample of virus was also frozen and thawed before and after incubation with neuraminidase to confirm that the Icell virus was no longer inactivated by freezing and thawing after neuraminidase treatment. Influenza virus was treated with Triton X-100 for 20 min at room temperature without prior purification. A. Sindbis virus grown in fibroblasts from I-cell disease patient L. T. (A)+neuraminidase, (O)-neuraminidase; Sindbis virus grown in fibroblasts 312 (A)+neuraminidase, (0)-neuraminidase. B. Sindbis virus grown in fibroblasts from I-cell disease patient T. M. (A)+neuraminidase, (O)-neuraminidase; Sindbis virus grown in fibroblasts 312 (A)+neuraminidase, (O)-neuraminidase. C. Influenza virus grown in fibroblasts from I cell disease patient L. T. (0); in tibroblasts S291 (0).

The most striking observation in these studies is that treatment of I-cell Sindbis virus with neuraminidase both protected the virus from inactivation by freezing and thawing and reactivated the virus after it had been inactivated. We had been led to this discovery by the finding that, in contrast to both Sindbis virus and VSV, influenza virus grown in I-cell fibroblasts was not more sensitive to freezing and thawing than was the virus grown in normal human fibroblasts. One way to explain these observations is to propose that Sindbis virus grown in I-cell fibroblasts has an increased number of sialic acid residues which confer freeze-thaw sensitivity on the virus. An equally plausible hypothesis is that some other component in the viral membrane is altered such that interaction with sialic acid leads to inactivation by freezing and thawing. In either case, treatment with neuraminidase could protect the virus from inactivation. Our data demonstrate that the glycopro-

NEURAMINIDASE

AND

I-CELL

SINDBIS

VIKUS

415

FIG. 3. Gel filtration of glycopeptides of Sindbis virus. Sindbis virus from I-cell fibroblasts was labeled with [“Clglucosamine, the virus from normal human fibroblasts was labeled with [‘Hlglucosamine. The virus preparations were mixed, treated with Pronase and an aliquot was chromatographed on a Bio-Gel P6 column (A) (Sly et al., 1976). A second aliquot was treated with neuraminidase (Keegstra et al., 1975) after the pH had been adjusted to 5.2 with acetic acid. This sample was then chromatographed on the same column (B).

teins of I-cell Sindbis virus have the same amount of sialic acid as the glycoproteins of virus obtained from normal human fibroblasts. Furthermore, an increase in the sialic acid content of virion glycoprotein does not confer freeze-thaw sensitivity on viruses such as VSV and influenza virus. When VSV was grown in an L cell variant that has a high level of sialyltransferase activity, the virion glycoprotein contained excess sialic acid (Gottlieb and Kornfeld, 1976). This virus showed the same stability to freezing and thawing as VSV from normal L cells (S. Schlesinger, unpublished results). (It was not possible to test Sindbis virus as it does not produce progeny in this variant (Gottlieb, Kornfeld, and Schlesinger, manuscript in preparation)). Sialic acid can be added to the hemagglutinin of influenza virus using sialyltransferase prepared from colostrum (Schulze, 1974). The stability of I-cell influenza virus to freezing and thawing was not affected by the addition of sialic acid to the virion glycoprotein (I. Schulze, unpublished observations). Although the glycoproteins of I-cell Sindbis virus are not hypersialylated, there may

be an increase in the level of sialic acid in I-cell viral glycolipids. Dawson et al. (1972) found that the glycolipids of I-cell fibroblasts contain 4 to 9 times more Go:{ (NANA-cu-(2+8)-NANA-u(2+3)-Gal-P(I-4) Glc-Cer) than glycolipids from normal human fibroblasts. Our preliminary studies show that the virus grown in human libroblasts has a more complicated glycolipid pattern than virus grown in chicken embryo fibroblasts (Hirschberg and Robbins, 1974). However, we have not yet obtained sufficient amounts of virions from normal human and I-cell fibroblasts to compare the sialic acid content of their glycolipids. I-cell fibroblasts were reported to be deficient in neuraminidase activity (Thomas et al., 1976; Cantz et al., 1977) and this could account for higher levels of sialic acid in some membrane components. Our finding that Sindbis virus grown in fibroblasts with a neuraminidase deficiency (mucolipidosis 1) is only moderately sensitive to freezing and thawing may reflect quantitative or qualitative differences in the severity of the neuraminidase defect. I-cell fibro-

416

SCHLESINGER,

SLY,

blast extracts had about 4-fold higher levels of sialic acid per milligram of protein than extracts of fibroblasts from mucolipidosis I patients (Cantz et nl., 1977). l-cell Sindbis virus is very sensitive to Triton X-100 and this property is not affected by neuraminidase. These results could be explained by the inabi1it.v of neuraminidase to remove those sialic acid residues critical to interaction with Triton X100, but they also suggest that there may be additional changes in the virus. The sensitivity of liposomes to Triton X-100 is greatly affected by both the phospholipid composition and the percentage of cholesterol (Inoue and Kitagawa, 1976). Thus, changes in these components could be responsible for the detergent sensitivity of lcell Sindbis virus. The only reason to suspect that sialic acid may be involved in the Trit,on X-100 sensitivity of l-cell Sindbis virus is that l-cell influenza virus shows the same degree of inactivation by Triton X100 as does the virus from normal human fibroblasts. However, influenza virus obtained from both cell types is inactivated by such low concentrations of Triton X-100 that changes in sensitivity due to changes in lipid composition may not, be detectable. The mechanism of inactivation of l-cell Sindbis virus by freezing and thawing remains unknown. Inactivation is not due to aggregation and is clearly reversible by treatment with neuraminidase. We can only speculate at this point that freezing of the l-cell virus causes some change in structure of the membrane which cannot be reversed by thawing unless the sialic acid residues are removed. One of the original goals in this study was to use an enveloped virus, such as Sindbis virus, to probe the structure of the membrane of I-cell fibroblasts. In spite of our inability to ident,ify the chemical defect in l-cell Sindbis virus, we have shown that the virus does acquire altered membrane components from the host. It may be possible to obtain a better understanding of the mechanism of inactivation by physical measurements of these “freeze“freeze-inactivated,” and sensitive,” “freeze - inactivated - neuraminidase - reactivated” virions. In this way, l-cell viral membranes may provide a useful tool to under-

AND

SCHULZE

stand the functional relationships of some normal membrane components.

This research was supported b,v the t’ollowittg grants: Al 11:177 (S. S.), GM “1096 (U’. S. S.). Al 10097 (I. ‘1’. S.). anti the Hanken do&n Trust f’or C’rippling I)israses in Children. We thank Nancy Gelh, Marjorie I’ttuling Ievthetr. trnd I&abeth Laguinsktt f’or their hcalp in cttrrying out many of’ theses experiments.

I)AWSON. G., MATAI.ON. K., anti DORFMAN. A. (1972). (~I\c~osl~hin~oliI,itls in c.ulturrtl hutxxtn skin l’ibroblasts. Characterizai ion and rnctaholistrr in l’ihroblasts from patients with inborn errors of glvco~ sphingolipiti and tnucoIt(,l\~sa~charidr metttholisnt. ,J. H/o/. ~‘/wt,r. 247, X151-5:15X. GOTTI.IKI~. C., ttntl KOK~FBI.I). S. (1976). 1sol;ction ctnd chara~teriztttion ol’ two tnousc I, cell lines rrsistant to the toxic lectin ricin. ./. Bid. (‘izc~t. 251, Xfil-ix-L

III(.KMAN. S.. SHAPIRO. I,. .J.. ttntl NR’:IJFEI.I). Cl. F. (1973). A rt~cognition tnarker required for uptake 01 a Iysosomal enzyme by culturetl fihrohlasts. Biochcm. Wzo~phys. Kcs. (‘ommun. 57, 55-E I. IIIHSt‘HHEKt:, c’. H., and IbHBIi%S, I’. W. (19741. ‘I‘hr glycttliltids and phosphrtlipicls 01’ Sindt)is vit,tt:, and their rrlutiun to (he lipids of the host cell plasma mctnbntne. Virol0g.v 61, WL-GOX. KAPI.AN. A., A~HOHI). I). T., and SI.U. W. S. (1975). I’hosphohrxosyl components ot’a I,vsosontal enryme are recognized by pinocytosis recy~tors on human fihrol~ltssts. I’loc. Nnt. Actrd. Sri. 1 %‘A 74, 3Efi-“fl:lo. KRRGSTHA. K.. SKFTON. B. M.. and HIJRK~, 11. (1975). Sindhis virus glycoproteins: effect of the host cell on the oligosaccharities. .I. Vtrol. 16: fjl:!-620. KEI.I.Y. ‘1’. IX.. and GRAETZ. G. (1977). Is&ted acid neurtuninidase tieficicncy: a distinct Iysosomal storage disease. .I. 1Mcrf. &ne/. 1, :I I-36. LRKOY, .I. G.. and I)EMAKS, I<. 1. (I!Jfi7). Mutant

NEURAMINIDASE

AND

I-CELL

SlNDBIS

417

VIRIJS

Mahv. eds.). pp. 161-1i5. Academic Press. New York. SEFTON, H. M.. and KEFXSTKA, K. ( 1974). Gl~coproteins of Sindhis virus: prrliminarg characterization of the oligosaccharides. ,I. Viral. 14, 5?2-530. SI.Y, W. S., LAGWINSKA, F:.. and SCHI~~INGKK. S. ( Illiti). Envelolwd virus acquired membrane defect when passaged in f’ihrohlaets from I-cell disease patients. I’tw. :Vcc/. Actrd. S’CI. 1 .X4 73, 2443-24.47. SYMINGTON. .J., and SCHLESINGEK. M. .J. ( 1955). ISOlation of a Sintlbis virus variant hy passage on mouse plasmacytoma cells. .I. \%,o/. 15, lO:lT-1041. THOMAS, G. H., TIIUX. G. E.. ,JK., H~~~,wr.rx+, I,. W., MII.I,EK. (1. S.. and HACT, .I. W t l%‘fi). Incrrascd Irvrls of sialic, acid associated with a sialidase tl& ciency in I-cell disease (mucolipitlosi.ioiipidosis 11) fibrohl;rsts. Hiochctn. HW/~h~,S. flus. (‘ommrm. 71, IH8-1%. VI,AI)I.TII’. G. I).. atrd RATTAZZI. M. C. (IS’iS). Atmormal l~sosomal h~drolases cwxw eti 1,~ cultured fibroblasts in I-cell disease tmucolipitlosis II). Hiodwn. Hioph~s. Rex (‘on~rnun. 67, Wi-964. ~VIESMANN.

biological ;ic’t ivit ies of the influenza viritrn. I,I “Ncgat i\ I’ St rantI Virusrs.” (It. I>. Hart-v and B. W. .I.

Il.

N.,

and

HKKSCHKOWIT~.

N.

Sludiw on the pat hogenetic mechanism tlise;se in cultured fihrohlasts. I’cvl~rrtr.. Xfi’,-s-0. t ,

LX. (1974).

of I-wll Kw. 8,