A temperature-sensitive mutant of Sendai virus with an altered hemagglutinin-neuraminidase polypeptide: Consequences for virus assembly and cytopathology

VIROLOGY

67, 179-187

(19751

A Temperature-Sensitive Altered

of Sendai

Hemagglutinin-Neuraminidase

Consequences ALLEN

PORTNER,

of Virology

and Cytopathology

RUTH A. SCROGGS, D. W. KINGSBURY

and Immunology, St. Jude Children’s Memphis, Tennessee 38101 Accepted

April

Virus with an Polypeptide:

for Virus Assembly AND

Laboratories

Mutant

P. A. MARX,’

Research

Hospital,

P. 0. Box 318,

28, 1975

Sendai virus mutant ts 271 was previously shown to be an RNA positive mutant with a temperature-sensitive hemagglutinin. We now report that noninfectious virus particles are produced when this mutant is grown at the nonpermissive temperature. These virus particles appear to have only one defect: they are devoid of a single polypeptide, the 70,000 dalton HN glycopolypeptide responsible for hemagglutinin and neuraminidase activities. Consequently, the noninfectious particles lack these activities and they are incapable of attaching to cells. Moreover, cells producing the particles contain neither hemagglutinin nor neuraminidase activities, suggesting that the relevant glycopolypeptide does not assume a functional conformation when it is synthesized under nonpermissive conditions. We conclude that Sendai virus morphogenesis does not require HN as a structural element or any function supplied by HN. Other consequences of the mutation were marked lessenings of cytopathology and cell protein synthesis inhibition during infections at nonpermissive temperature, indicating that the native HN glycopolypeptide is an important factor in cell killing by Sendai virus. INTRODUCTION

Among the seven complementation groups to which Sendai virus ts mutants have been assigned so far, only one has no apparent defect in viral RNA synthesis (Portner et al., 1974). This complementation group is represented by a single mutant, ts 271. Virions of this mutant agglutinate erythrocytes normally at 30”, but not at 38”, the nonpermissive temperature for virus growth. We have localized the ts 271 defect in the virion glycoprotein fraction, and we assume that the HN glycopolypeptide, the one which is responsible for the hemagglutinin and neuraminidase activities of Sendai virus and which migrates with an apparent molecular weight of 70,000 on ‘Present address: Department of Microbiology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107. 179 Copyright@ All rights

1975 by Academic Press. Inc. of reproduction in any form reserved.

SDS-acrylamide gels (Tozawa et al., 1973; Scheid and Choppin, 1974), contains the mutation. Other paramyxoviruses have been shown to contain a glycopolypeptide with similar properties (Scheid et al., 1972; Scheid and Choppin, 1973). In this paper we report some consequences of this ts 271 mutation for the morphogenesis of Sendai virus and the cytopathology it produces. MATERIALS

AND

METHODS

Viruses. Our wild-type Sendai virus and the origin of mutant ts 271 have been described (Portner et al., 1974). For the experiments reported here, viruses were propagated and titrated for infectivity in chicken embryo lung (CEL) cell monolayer cultures (Darlington et al., 1970). To label virus particles, infected cells were incubated with 3H- or “C-labeled amino acid

180

PORTNER

mixtures or with [3H]uridine for 48 hr at 30” or 38” starting at 24 or 48 hr after infection. Enzyme assays. Virion transcriptase reactions were performed for 5 hr at 24” (Stone et al., 1971). Neuraminidase was measured at 30” (Portner et al., 1974). Labeling of polypeptides in infected cells. Infected cells were incubated at 30” or 38” for various times as indicated in Results. The medium was then replaced by Earle’s saline and incubation was continued for 30 min to deplete amino acid pools. Thereafter, labeling was for 1 hr with 25 &i of [3H]amino acid mixture (Schwarz BioResearch) per ml of Earle’s saline. Cells were then scraped from the plates, precipitated with 5% trichloracetic acid, washed with acetone, and dissolved in electrophoresis sample buffer as described below. Polyacrylamide gel electrophoresis. An SDS-urea-7.5% acrylamide gel system was used (Zweerink et al., 1971), affording enhanced resolution of polypeptides greater than 60,000 MW. Labeled virus particles or infected cells were dissolved by boiling for 2 min in 1% SDS, 1% mercaptoethanol, 5 M urea, 0.1% bromphenol blue, and were applied in a volume of 50 ~1 or less. A potential difference of 40 V was applied for 18 hr; gels were then sliced and counted (Stone et al., 1972). RESULTS

Production of noninfectious ts 271 virus particles. Our previous work showed that no hemagglutinating or infectious material was produced when cells infected by ts 271 were incubated at 38” after 30 min adsorption at 24” (Portner et al., 1974). But this did not exclude the possibility that virusrelated materials lacking these activities were released into the medium. To test this, ts 271-infected cells were labeled with [3H]amino acids at 38” and culture fluid was centrifuged to equilibrium in a D,Osucrose density gradient. Figure 1 shows that labeled particles were produced which banded at a density slightly less (1.155 g/ems) than wild-type virions (1.164 g/ cm3). Electron microscopy (performed by Dr. R. W. Darlington) revealed virus parti-

ET AL.

6

7 0 x % 3v

0

I loo

1150

DENSITY

I.200

1.250

v 0

0

(gm /cm3)

FIG. 1. Isopycnic centrifugation of ts 271 particles made at 38”. At 24 hr after infection [‘HIamino acid mixture was added at 25 &i/ml and 48 hr later the medium was removed. The medium was centrifuged for 10 min at 3600 g. [“C]amino acid labeled wildtype virions were added to the supernatant fluid and the mixture was layered on a linear DpO-sucrose gradient (1.15-1.33 g/cm!‘) containing 0.005 M Tris-HCl, 0.001 M EDTA (pH 7.4). The preparation was centrifuged for 16 hr, 12” at 20,000 rpm. Symbols: (W) ts 271,38”; (0- - -0) wild-type virus, 38”.

cles which were grossly similar in appearance to wild-type virus. Yields of noninfectious particles were never less than 50% or more than 200% of wild-type virus yields, as measured by incorporation of radioactive amino acids or uridine into the particles or by their protein contents (Lowry et al., 1951). The density difference in Fig. 1 appears to be characteristic of the mutant virus particles made at 38”, because ts 271 virions made at 30” banded at the same density as wild type. Another characteristic was increased fragility. Attempts to recover these particles in a high-speed pellet were unsuccessful, whereas wildtype virions and ts 271 grown at 30” survived this treatment. As expected, the mutant virus particles

SENDAI

VIRUS

made at 38” and purified in D20-sucrose gradients were noninfectious and did not hemagglutinate. In addition, they exhibited little or no neuraminidase activity against a fetuin substrate (Table 1). Furthermore, they were incapable of attaching to susceptible cells (Table 1) and they were devoid of fusing or hemolytic activities (data not shown). RNA and transcriptase in the noninfectious particles. We examined noninfectious

ts 271 particles produced at 38” for 50s RNA by labeling cells with [3H]uridine from 48 to 72 hr after infection and centrifuging the culture fluids in SDS-containing sucrose gradients. The noninfectious particles contained about as much as 50s RNA as wild-type virus or ts 271 produced at 30” (data not shown). The noninfectious particles contained normal amounts of Sendai virus nucleocapsid polypeptides (Fig. 2). In vitro assays of RNA dependent RNA polymerase (transcriptase) revealed especially abundant activity in these particles (Table 1). The activity was consistently sevenfold greater than in infectious ts 271 or wildtype virus. Polypeptides in noninfectious ts 271 particles. To this point, the evidence indicated

that ts 271 particles made at 38” contained intact RNA genomes and a functional

transcriptional apparatus. They appeared to be noninfectious because they lacked surface elements essential for attachment to susceptible cells. Polyacrylamide gel electrophoresis of the particles revealed the nature of the defect: the HN polypeptide was absent (Fig. 2A). All other virion polypeptides were present in the usual proportions. The heterogeneous material migrating in the upper portion of the gel (fractions l-35, Fig. 2A) was absent from wild-type virions. It seems unlikely that it is aberrant HN, because it is so abundant. It may represent cell polypeptides incorporated into virions or adsorbed to them. The latter possibility arises because viral neuraminidase may prevent sialic acid-mediated binding of glycoproteins to virus particles (Palese et al., 1974). When ts 271 was made at permissive temperature, the virus polypeptide composition was altered slightly. HN was present, but in reduced amounts, and there was some heterogeneous material at the top of the gel (Fig. 2B). The reduction in HN correlates with reduced neuraminidase activity compared to wild type, even at permissive temperature (Table 1; Portner et al., 1974). A noteworthy feature of the SDS-urea gels used here was a clear separation of HN

TABLE PROPERTIES Virus

ts 271 ts 271 Wild type

Growth temperature”

38" 30" 38"

181

MUTANT

OF ts 271

1

VIRUS PARTICLES

PFU per cultureb

HA units per cultureb

1.2 x 103 3.6 x IO9 6.0 x lo8

<3x IO' 2 x 103 1.5 x 103

Neuraminidase &dmg protein/hr)’

0.42 3.75 11.1

Tran-

scriptase

9% Attached to cells’

wdmg proteind

41,050 6220 6175

0 16 24

a CEL cells were infected at an input multiplicity of 1 PFU per cell, held at 24” for 30 min, and incubated at the indicated temperature for 48-72 hr. Virus particles were isolated from the medium by isopycnic centrifugation as described in the legend of Fig. 1. b Infectivity and HA assays were performed on the culture media before isopycnic centrifugation. c Neuraminidase was assayed at 30”. d Acid-insoluble SH-GMP incorporated in 5 hr at 24”. e This is the fraction of [SH]amino acid-labeled virus particles which were cell-associated after 2 hr at 24” followed by four washes with phosphate-buffered saline at 4’. The input multiplicity was about 50 PFU per cell of wild-type virus or ts 271 made at 30”. In the case of ts 271 made at 38”, an equivalent number of counts (20,000) was used.

182

PORTNER I

6-

ET AL. I

I

PHNFoNP

F

A

1

I P HN

B

I

Fa NP

1111

F 1

20

h 0 r”

K)

25

50

FRACTION

75

II

NUMBER

FIG. 2. Polyacrylamide gel electrophoresis of polypeptides in ts 271 virus particles. [3H]amino acid labeled virus particles made at 38” or 30” were isolated by isopycnic centrifugation as in Fig. 1 and electrophoresed in SDS-urea gels as described in Materials and Methods. In each case, [“‘Clamino acid labeled wild-type virion polypeptides were run in the same gel. The direction of migration is from left to right. Virion polypeptides are marked as suggested by Scheid and Choppin (1974). Panel A: 3H-ts 271, 38”. Panel B: 3H-ts 271, 30”.

from a second component which moved slightly behind NP (fraction 50, Fig. 2). We have designated this component FO, on the assumption that it is the same polypeptide considered to be a high molecular weight precursor of the fusion factor glycoprotein, F (Homma and Ohuchi, 1973; Scheid and Choppin, 1974). We always see it in fully infectious virus produced in CEL cells. Presumably, not all of the F, is cleaved to F in CEL cells. Similar observations have been made by Lamb and Mahy (1974). Changes in ts 271 infected cells-HN actiuities. The preceding results showed

that the HN polypeptide does not exist in its normal form in ts 271 particles made at 38”. It was therefore of interest to inquire

whether either hemagglutinin or neuraminidase activities appeared in any measurable form in infected cells. Intact cells producing noninfectious particles at 38” were tested for hemagglutinating moieties on their surfaces by a hemadsorption assay. The results were negative (Table 2). Cells were also sonicated to release hemagglutinin which might exist in intracellular forms, such as in “viromicrosomes” (Rott et al., 1963). Again, no agglutinating activity was found. Since endogenous neuraminidase activity is low in CEL cells, this enzyme was measured in sonicated infected cells. As shown in Table 2, ts 271-infected 38” cells revealed scant neuraminidase activity, whereas neuraminidase

SENDAI

VIRUS TABLE

VIRUS-SPECIFIC Virus ts 271 ts271 Wild type

Incubation temperaturea 38” 30” 38”

ACTIVITIES

Hemadsorption %b o-3 95-100 95-100

183

MUTANT 2 IN ts 271-INFECTED H~Ounirisper 6 e <30 270 810

CELLS Neuraminidase (A,,$mg protein/hrY 0.36 1.80 5.88

a CEL cells were infected as described in Table 1 and incubated at the indicated temperature for 72 hr. b Percentage of cells with one or more erythrocytes attached after incubation at 4’ for 30 min with a 1% suspension of chicken erythrocytes in phosphate-buffered saline, followed by four washes with phosphate-buffered saline at 4”. c Cells were suspended in phosphate-buffered saline and disrupted by sonication for 1 min. d Cells were suspended in 0.2 M Na phosphate (pH 5.9) containing 2% Triton X-100 and sonicated for 1 min. Neuraminidase was assayed at 30”. The neuraminidase activity of uninfected cells (grown at 38”) was 0.15 A,,,lmg protein/hr.

was readily detected in cells making ts 271 at permissive temperature and in cells making wild-type virus. These results indicate that at nonpermissive temperature the ts 271 defect either blocks the synthesis of HN or prevents its assumption of a functional conformation.

reduced to about half the level obtaining at nonpermissive temperature, and HN was readily detected (Fig. 3C). A corollary of these findings was the microscopic appearance of the cells. If kept at 38”, ts 271-infected cells could be maintained free of cytopathic effects for weeks, and they were even successfully subculChanges in ts 271-infected cells-protein sypthesis. In an attempt to learn if the HN tured twice. At 30”, ts 271 infection depolypeptide was synthesized at nonpermisstroyed the cells by 72 to 96 hr, as does sive temperature in ts 271-infected cells, wild-type infection. we separated cell polypeptides labeled for 1 Thus, it appears that the HN polypephr at 48 hr after infection, a time when tide plays an important role in Sendai viral protein synthesis is clearly visible virus-induced cytopathology. against a reduced background of cellular DISCUSSION protein synthesis (Stone et al., 1972; Lamb and Mahy, 1974). Unfortunately, cellular We think it is likely that the only defect protein synthesis was not decreased suffi- in Sendai virus mutant ts 271 is in the HN ciently during ts 271 infection at 38” to polypeptide. In our previous paper (Portunmask virus-specific polypetides and per- ner et al., 1974), we showed that ts 271 mit any deductions, especially in the re- grown at permissive temperature had a gion of interest, where HN migrates (Fig. temperature-dependent hamgglutinin and 3A, fraction 35). Thus we cannot exclude this behavior was exhibited by a glycopolythe possibility that ts 271 HN synthesis is peptide fraction isolated from the mutant blocked at 38”. virions. This almost certainly places the On the other hand, the failure of cellular defect in the HN polypeptide itself. At that protein synthesis to decline in ts 271- time, we also showed that ts 271-infected infected cells at 38” is of interest in itself. cells made both kinds of virus-specific As the remaining panels in Fig. 3 show, this RNA (genomes and transcriptsj at nonperfailure is a temperature-dependent phe- missive temperature. We now have evinomenon. Cellular protein synthesis is dence that all viral proteins (with the clearly inhibited in ts 271 infection after 48 possible exception of HN) are made in ts hr of incubation at 30” (Fig. 3B), and the 271-infected cells at nonpermissive teminhibition sets in rapidly when cells are perature, because virus particles are proshifted down from 38” to 30” (Fig. 3C, D). duced in about normal amounts under Indeed, by 2 hr after shift to permissive these conditions and the only structural temperature, cellular protein synthesis was element these virus particles lack is HN.

184

PORTNER

ET AL.

The simplest explanation for the absence of HN in these particles is that the mutation interferes with the assumption of the normal (functional) conformation of the polypeptide when it is synthesized at nonpermissive temperature. When ts 271 HN is made at permissive temperature and incorporated into virions the defect is reversible; the mutant virions can repeatedly be made to agglutinate erythrocytes or fail to agglutinate them by simply shifting the temperature from 30” to 38”. But when the ts 271 HN polypeptide is made at nonpermissive temperature, no hemagglu-

II

A

2 30 a u ,I

0 t

P HN

tinin or neuraminidase activity can be measured at either temperature after the cells are disrupted. The implication is that any or all of the steps involved in the maturation of HN as a surface membrane glycoprotein (insertion into an intracellular membrane, transport to the surface, glycosylation, proteolytic processing) cannot take place if the correct initial conformation does not form, but once all these things have happened, the finished product is stabilized against irreversible inactivation by a temporary shift to nonpermissive conditions.

FoNC

F

11

1

P HN FoNc

F

11

11111

FRACTION

T

NUMBER

FIG. 3. Polyacrylamide gel electrophoresis of polypeptides from cells infected by ts 271. Virus was added to CEL cells and after 30 min at 24” the cultures were placed at 30” or 38” for 48 hr. Some cultures were labeled immediately with [sH]amino acids. Others were shifted from 38” to 30” and maintained at 30” for various times before labeling. After 1 hr of labeling the cells were scraped from the plates and prepared for electrophoresis as described in Materials and Methods. In each case, [‘CJamino acid labeled wild-type virions were run in the same gel. Panel A: 38” continuously. Panel B: 30” continuously. Panel C: 2 hr after shift-down from 38” to 30”. Panel D: 24 hr after shift-down from 38” to 30”.

185

SENDAI VIRUS MUTANT ,

,

I

-6 P HN

FoNC

1111

F

M

1

1

& 3z P HN Fo NC

1111

FRACTION FIG.

F

6

1

v 0

1

NUMBER

8-Continued

Much less likely, in view of ease of complementation and a reasonable reversion frequency (Portner et al., 1974), is the possibility that ts 271 is a double mutant with one defect in the HN polypeptide and a second in a function required for the production of a functional HN, although we have not formally ruled this out. The consequences of the absence of HN from the noninfectious particles were more or less predictable. The loss of both hemagglutinin and neuraminidase activities from the particles together with the loss of this single polypeptide is a satisfying confirmation of the work of others (Scheid et al., 1972; Scheid and Choppin, 1973, 1974; Tozawa et al., 1973). Failure of these particles to attach to host cells was worth documenting, because hemagglutination

may require especially strong virus-cell binding, the viral receptors of host cells may not be identical to those of erythrocytes, and the remaining glycopolypeptides in the noninfectious particles, especially F, might have mediated attachment. The results show that HN is essential for viruscell binding, either as a required structure, or because of its neuraminidase activity (Palese et al., 1974) or both. Tests of hemolysis and cell fusion were, of course, negative, in consequence of the failure of the noninfectious particles to attach to cells. The fact that virus particles could be produced without HN has important implications. It shows that paramyxovirus morphogenesis is independent of HN either as a structural element or as a supplier of

186

PORTNER

neuraminidase activity. Indeed, the absence of virus-induced neuraminidase in the cells at nonpermissive temperature indicates that this enzyme plays no role in any virus-specified event between eclipse and the release of progeny from the cell. The report of noninfectious paramyxovirus particle production in the presence of 2-deoxy-D-glucose (Hodes et al., 1975) provides an instructive parallel to our findings. The targets of this inhibitor are the viral envelope glycoproteins, and it seems likely that the production of particles devoid of surface projections is a reflection of a block in viral surface glycoprotein insertion into the viral envelope (Hodes et al., 1975). Unexpected and remarkable was the evidence that the HN polypeptide plays a leading role in virus-induced cytopathology. It will be important to learn the mechanism by which HN acts to turn off cell functions. The nature of HN suggests that it is acting at the membrane level, either by virtue of its neuraminidase activity (What are the effects of prolonged neuraminidase action on cell membrane proteins?) or perhaps by modifying the membrane in which it is inserted in a different way, such as by replacing functional cell proteins. Finally, the implications of the elevated transcriptase activity in the virus particles lacking HN may be considered. Previously, we showed that a glycoprotein fraction of Sendai virus particles inhibited the nucleocapsid-associated transcriptase in vitro (Marx et al ., 1974). The high transcriptase activity in the present case may simply reflect the absence of the inhibitor, HN. We suggested that the inhibition of nucleocapsid transcription by envelope proteins might be a factor in virus assembly (Marx et al., 1974). In the case of HN, this possibility now seems to have been ruled out. ACKNOWLEDGMENTS Andrew Moseley provided skilled technical assistance. We thank Exeen Morgan for helpful advice. This work was supported by USPHS Research Grants AI-05343 and AI-11949, USPHS Cancer Research Center Grant CA-08480, USPHS Research Career

ET AL. Development Award bury, and by ALSAC.

HD-14,491

to David

W. Kings-

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SENDAI VIRUS MUTANT infected cells: polypeptides in the transcriptive complex. J. Viral. 10, 1037-1043. STONE, H. O., PORTNER, A., and KINGSBURY,D. W. (1971). Ribonucleic acid transcriptases in Sendai virions and infected cells. J. Viral. 8, 174-180. TOZAWA, H., WATANABE, M., and ISHIDA, N. (1973). Structural components of Sendai virus. Serological

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and physicochemical characterization of hemagglutinin subunit associated with neuraminidase activity. Virology 55, 242-253. ZWEERINK, H. J., MCDOWELL, M. J., and JOKLIK, W. K. (1971). Essential and nonessential noncapsid reovirus proteins. Virology 45, 716-723.