A mutant of rous sarcoma virus with a conditional defect in the determinant(s) of viral host range

VIROLOGY

i7,

443-456

(1977)

A Mutant of Rous Sarcoma Virus with a Conditional Determinant(s) of Viral Host Range WILLIAM The Institute

for

Cancer

S. MASON’

Research,

The Fox Accepted

Chase

AND

CAROL

Cancer

Center,

November

Defect in the

YEATER Philadelphia,

Pennsylvania

19111

I, 1976

A temperature-sensitive mutant of the Prague strain of Rous sarcoma virus of subgroup C, tsPH734PR-C, replicates much less efficiently at the nonpermissive (41”) than the permissive (35”) temperature while transforming equally well at both temperatures. In contrast, the wild-type parent, urtPR-C, is able to replicate and to transform chick embryo fibroblasts equally well at both 35” and 41”. Two lines of evidence suggest that tsPH734PR-C is defective in the synthesis or utilization of virus envelope glycoproteins GP85 and GP35. First, tsPH734PR-C appears to be defective in the incorporation of the virus envelope glycoproteins GP85 and GP35 into the noninfectious virus particles synthesized at 41”. Second, tsPH734PR-C, a host range subgroup C virus, is not complemented for replication of subgroup C virus at 41” by coinfection of cells with RAV-6 avian leukosis virus of subgroup B or by coinfection with the defective Bryan strain of Rous sarcoma virus, BH-RSV( -).

group C. This mutant is designated tsPH734PR-C. The data which we present are most consistent with the interpretation that tsPH734PR-C has a conditional temperature-sensitive defect in the viral glycoproteins GP85 and/or GP35. Tato, Beamand, and Wyke (personal communication) have recently obtained evidence that tsLA30mPR-A (Wyke and Linial, 1973) also contains a mutation affecting GP85 or GP35. While tsPH734PR-C is a late replication mutant, tsLA30mPRA is an early-coordinate mutant defective in initiation of infection at the nonpermissive temperature (Tato et al., personal communication).

INTRODUCTION

While several mutants of Rous sarcoma virus have been isolated that have temperature-sensitive alterations in functions required late in viral replication (Toyoshima and Vogt, 1969a; Friis and Hunter, 1973; Wyke, 1973; Wyke and Linial, 1973; Friis et al., 19751, in only a few instances has the altered gene product been apparent. Friis et al. (1975) showed that the mutant tsLA672 of the Prague strain of Rous sarcoma virus was unable to incorporate reverse transcriptase activity into virions at the nonpermissive temperature. More recently, Rohrschneider et al. (personal communication) and Hunter et al. (1976) showed that the B77 avian sarcoma virus mutant, tsLA334, possesses a temperature-sensitive lesion in PR76, the precursor to the major internal proteins of the virus (Vogt et al., 1975). We now report the isolation and characterization of a mutant of the Prague stain of’Rous sarcoma virus of subgroup C with a conditional temperature-sensitive defect in specification of viral host range of sub1 Address

reprint

requests

to Dr.

MATERIALS

AND

METHODS

Cells. Chick embryo fibroblasts were prepared from lo- to 11-day embryos of white Leghorn chickens and grown in Ham’s FlO medium supplemented with 10% tryptose phosphate broth, 10% calf serum, and 2% heat-inactivated chicken serum (PGM; Vogt, 1969). Primary cells were tested for chick helper factor as previously described (Weiss et al., 19731, and only chf-negative cells were used for clon-

Mason. 443

Copyright All rights

Q 1977 by Academic Press, of reproduction in any form

Inc. reserved.

ISSN

0042-6622

444

MASON AND YEATER

ing and growth of virus. C/B cells infected with the defective Bryan -high-titer strain of Rous sarcoma virus [BH-RSV(-)I were provided by Drs. Young Chen and Peter K. Vogt, University of Southern California School of Medicine, Los Angles, California. The BH-RSV(-)-infected cells were maintained in cloning medium (Duesberg and Vogt, 1973), and this medium was changed twice daily. Viruses. The wild-type virus was the Prague strain of Rous sarcoma virus of subgroup C (wtPR-C). The temperaturesensitive mutant tsPH734PR-C was isolated from z&PR-C grown in the presence of 5-azacytidine (Toyoshima and Vogt, 1969a). The permissive temperature for the mutant is 35”, while the nonpermissive temperature is 41”. Two other temperature-sensitive mutants of wtPR-C were used in this study, the reverse transcriptase mutant ts LA337PR-C defective in the initiation of infection at 41” (Linial and Mason, 1973) and the coordinate mutant tsLA338PR-C (Wyke and Linial, 1973; Hunter and Vogt, 1976) defective in cell transformation and virus replication at 41”. The avian leukosis virus RAV-6 belongs to avian tumor virus subgroup B. wtLA337:RAV-6-B was isolated as a subgroup B wild-type RSV recombinant between tsLA337-C and RAV-6 (Mason et al., 1974). Rous sarcoma virus-infected cells were grown in Ham’s FlO medium supplemented with 10% tryptose phosphate broth, 5% calf serum (GM), and 1% dimethyl sulfoxide (DMSO) from 1 day postinfection (Vogt et al., 1970). Cells were infected and transferred in medium supplemented with 10% tryptose phosphate broth, 5% calf serum, and 2 pglml of Polybrene (Aldrich Chemical Company, Milwaukee, Wisconsin) to enhance the absorption and spread of virus to uninfected cells (Toyoshima and Vogt, 1969b). PGM replaced the DMSO medium in the growth of uninfected or RAV-ginfected cells. Tissue culture media from infected cells were harvested ca. 24 hr after a medium change and stored at -90” as a source of infectious virus.

Abbreviated designations for the viruses used in this study are consistent with the proposals of Vogt et al. (1974) and are used for convenience of recognition. Virus assays. Focus assays on chf-negative chick embryo fibroblasts were performed according to standard procedures (Vogt, 1969; Mason et al., 1974). Unless noted, focus assays were done at 35”. Concentrations of infectious sarcoma virus are expressed in FFU (focus-forming units) per milliliter. Isolation of transformation-defective derivatives of Rous sarcoma virus. To isolate a transformation defective (td) mutant of tsPH734PR-C, this virus was first assayed in the focus assay. Nontransformed areas of cells in the vicinity of foci were aspirated with a capillary pipet and transferred to a susceptible monolayer of chf-negative chick embryo fibroblasts. The cells were then transferred several times until the monolayer became positive for the groupspecific (GS) antigen of avian tumor viruses as detected in the complement fixation assay using pigeon antiGS serum (Suzawa et al., 1966; Sarma et al., 1969; Bonar et al., 1972). The virus-containing tissue culture fluids were then harvested for further testing and isolation of transformation-defective virus from residual transforming virus. Radioactive labeling of viruses and cells. Cells were labeled with either a U14C-labeled L-amino acid mixture, L[U’4Clserine (153 mCi/mmol), L-[63Hlfucose (13.4 Ci/mmol), or D-[S3H(N)lglucosamine (10.13 Cihnmol) obtained from New England Nuclear, Boston, Massachusetts. Labeling with radioactive amino acids or a mixture of radioactive amino acids and radioactive fucase or glucosamine was performed in 199 medium (Morgan et al., 1950) containing either no amino acids or 2% of the normal concentration of amino acids, L-glutamine, 5% calf serum, and, where indicated, 1% DMSO. Labeling with a mixture of radioactive serine and glucosamine was done in Ham’s FlO medium containing 2% the normal amount of serine and 5% calf serum. Twelve to sixteen hours after addition of the radioactive precursor, the petri dishes

HOST

RANGE

MUTANT

were placed on ice, the media were harvested and chilled to 4”, and the virus was immediately purified at 4”. The cell monolayers were washed three times with 10 ml of cold PBS containing 1% DMSO and stored at -80”. Purification of virus. Radioactive tissue culture fluids were clarified for 15 min at 5000 rpm in the Sorvall HB-4 rotor, 100 pg of unlabeled wtPR-C (purified) was added as carrier, and the virus was centrifuged in a Spinco SW 50.1 rotor for 40 min at 30,000 rpm onto a cushion of 65% (w/v) sucrose in standard buffer (0.1 M NaCl, 0.01 M TrisCl, 0.001 M EDTA, pH 7.4). The viruscontaining fractions were diluted with standard buffer to lower the sucrose concentration and layered onto a 7.1-ml linear 15-50% (w/v) sucrose gradient containing standard buffer. The virus was then centrifuged to equilibrium for 16 hr at 24,000 rpm in a Spinco SW 40 rotor, and the gradients were collected into ca. 0.30- to 0.35ml fractions. The radioactive virus-containing fractions were then pooled and diluted at least lo-fold with standard buffer, and the virus was pelleted by centrifugation for 60 min at 40,000 rpm in the SW 40 rotor. Radioactivity in virus fractions was determined by adding aliquots to 0.05 mg of bovine serum albumin in a volume of 0.2 ml and precipitating with 1.0 ml of 10% (wl v) trichloroacetic acid (TCA) onto GFA filters (Whatman). The filters were washed several times with 5% (w/v) TCA, then with 95% ethanol and dried under a heat lamp. The dried filters were placed in scintillation fluid (42 ml of New England Nuclear Liquifluor per liter of toluene) and counted in a Beckman liquid scintillation spectrometer. All counts are corrected by subtraction of crossover and background. Immune precipitation. Specific rabbit antiserum to the purified GP85 of wtPR-C was provided by Dr. Dani Bolognesi, Duke University, Durham, North Carolina, To prepare cell lysates, 2 ml of lysis buffer [l% (v/v) Nonidet P-40, 1% (w/v) sodium deoxycholate, ‘0.05 M NaCl, 0.02 M Tris-Cl, pH 7.41 was added to the washed monolayer-s described above. After 15-min incubation at 4”, the total material from

OF

445

RSV

the petri dish was transferred to a 15-ml Corex centrifuge tube, mixed for 30 set using a Vortex mixer, and then clarified 15 min at 5000 r-pm in a Sorvall HB-4 rotor. The supernatant was clarified a second time at 30,000 rpm for 60 min in a Spinco No. 65 fixed-angle rotor. Immune precipitation was then carried out on the second supernatant. The procedure for indirect immune precipitation using 0.02 ml of anti-GP85 serum or normal rabbit serum and an excess of goat anti-rabbit y-globulin serum (Antibodies Incorporated, Davis, California) was as described by Halpern et al. (1974). Immune precipitates were then solubilized as described (Halpern et al., 1974), and O.l-ml aliquots were analyzed by electrophoresis on polyacrylamide gels. Electrophoresis

on polyacrylamide

gels.

Electrophoresis on 10% polyacrylamide gels was essentially as described by Laemmli (1970). Following electrophoresis, the separation gels were frozen to -80” and then sliced into 2-mm transverse sections. Eight milliliters of scintillation fluid [containing 42 ml of New England Nuclear Liquifluor and 70 ml of protosol:H,O (9:l) in a total volume of 1 liter in toluenel was added to each slice in a glass scintillation vial, the sealed vials were incubated 2-4 days at 50”, and they were then counted in a Beckman scintillation spectrometer. Materials. C/E and C/BE chick embryos were obtained from Heisdorf and Nelson Laboratories, Redmond, Washington; Japanese quail embryos for the chf test were from Truslow Farms, Inc., Chestertown, Maryland. C/C chick embryos of the Regional Poultry Research Laboratory inbred line 15 were provided by Dr. L. Crittendon, Regional Poultry Research Laboratory, East Lansing, Michigan. RESULTS

tsPH734-C Is Temperature Virus Replication

Sensitive

In

The biological properties of tsPH734-C are shown in Table 1 in comparison with wtPR-C and two other temperature-sensitive mutants of wtPR-C. tsPH734-C forms

446

MASON

AND

foci equally as well at 41” as does the wildtype parent wtPR-C from which tsPH734-C was isolated, indicating that tsPH734-C is able to initiate infection at the nonpermissive temperature (Table 1, column 2). By contrast, tsLA337-C, the reverse transcriptase mutant, forms foci at 41” with l/loo the efficiency of &PR-C or tsPH734-C, while tsLA338-C forms foci at 41” with less than Vlo,ooo the efficiency of wtPR-C or tsPH734-C . The wild-type virus is also seen to produce infectious progeny equally well at 41” or 35” (Table 1, column 3). In contrast, tsPH734-C produces progeny 100 times less efficiently at 41”, while tsLA337-C and tsLA338-C replicate at least 1000 times less efficiently at 41”. Thus, tsPH734-C is more leaky at 41” than either tsLA337-C or tsLA338-C but still quite defective when compared to wtPR-C. Since tsPH734-C is able to initiate infect& at 41”, the defect in replication must occur late in the infectious cycle. In the following section, we demonstrate that in uiuo complementation at 41” between RAV-6 avian leukosis virus of subgroup B and tsPH734-C results in the production of sarcoma virus of subgroup B, but not C, suggesting that tsPH734-C is defective in determining virus host range at 41”. TsPH734-C Is Not Complemented for Replication by RAV-6 Avian Leukosis Virus

The results of complementation between tsPH734-C and RAVS are presented in Table 2. Replication on C/E cells is shown in columns 3-5, while columns 6-8 show replication on C/E cells preinfected with RAV-6. Three types of cells are used to assay transforming virus produced by these cells, C/E cells susceptible to virus with either a subgroup B or subgroup C host range, C/BE resistant to virus with subgroup B host range, and C/C resistant to virus with subgroup C host range. The focus assays done on C/E cells (Table 2, columns 3 and 6) reveal that replication of sarcoma virus was more efficient at 41” on cells p&infected with RAV-6 than

YEATER TABLE MUTANTS Virus (1) wtPR-C tsPH734-C tsLA337-C tsLA338-C

OF

wtPR-C Titer

1

Rous (41”/35”jb

(2) 0.3 0.3 0.003 ca. 0.00001

SARCOMA

VIRUS”

Replication (41”/35”)’ (3) 0.84 ca. 0.01 ca. 0.001 5 0.001

a Replication data were selected from Table 2. For details, see footnotes to Table 2. b Titration of RSV grown at 35” in the focus assay at 35 and 41”. The ratio of FFU per milliliter at 41” to FFU per milliliter at 35” is given. c See footnotes to Table 2. Replication was on C/E chick embryo fibroblasts at 35 and 41”. The ratio is presented.

on uninfected cells and that the greatest increase in efficiency was obtained with the temperature-sensitive mutants. RSV production following LutPR-C infection, at 41” relative to 35”, is enhanced 8-fold on RAV-6 preinfected cells, perhaps indicating that a mixture of wtPR-C and RAVS grows faster at 41” than LutPR-C alone. In contrast, the 41 to 35” RSV production of tsPH734-C on RAV-6 infected cells is enhanced more than loo-fold and the RSV production of tsLA337-C or tsLA338-C on RAV-6 infected cells lOOO-fold. These results indicate that the transforming function of the subgroup C replication mutants can be encapsidated into infectious virus produced by RAV-6 preinfected cells at the nonpermissive temperature. To determine whether viruses with the subgroup C host range specificity were produced, the replication harvests were titered on C/BE cells resistant to RSV with the subgroup B host range. The results in Table 2 (columns 6-8) reveal that complementation with RAV-6 of subgroup B results in the production of subgroup C virus at 41” when the superinfecting virus is wtPR-C, tsLA337-C, or tsLq338-C but not when the superinfecting virus is tsPH734-C. Thus, tsPH734-C is defective in the ability to produce a functional subgroup C host range determinant in RAV-6 infected cells. The complementation of tsLA337-C by RAV-6 for replication at 41” was surprising since tsLA337-C was not complemented for

HOST

RANGE

MUTANT TABLE

IN VZVO COMPLEMENTATION Virus (1)

wtPR-C

tsPH734-C

tsLA337-C

tsLA338-C

wtLA337-RAV-6-B

Replication temperature 03 (2) 35 41 41135 35 41 41135 35 41 41135 35 41 41/35 35 41 41135

Replication C/E (3) 5 x 103 4 x 103 0.8 3.2 x 102 0 SO.01 1.5 x 10’ 0 50.0001 1.5 x 10’ 0 %0.0001 1.5 x 106 8 x lo6 5.3

OF tsPH734-C on C/E,

OF

2 BY AN AVIAN

focus

assaysb

C/BE (4) 3.7 x 103 3.1 x 103 0.84 1.5 x 102 0 SO.01 6 x lo3 2 ca. 0.001 8 x lo3 0 ~0.0001 0 0 -

447

RSV

c/c (5) 0 0 0 0 Not

tested

Not

tested

Not

tested

LEUKOSIS Replication

VIRUS.

C/BE (7)

C/E

6)

1 x 105 6 x lo5 6 1 x 105 1.6 x lo5 1.6 2.2 x 105 2.1 x 104 0.1 4.5 x 105 I x 104 0.16 0 0

RAV-6-B”

on C/E + RAV-B-B, assaysc

1.9 x 104 1.5 x 105 7.9 1.8 x lo3 34 0.02 8 x lo4 I x 103 0.09 8 x 104 9 x 103 0.11 0 0

focus c/c (8)

2.3 2.3

x x 10 2.8 x 4.1 x 1.5

lo4 lo5 lo4 104

Not

tested

Not

tested

Not

tested

a C/E chick embryo tibroblasts were infected with RAV-6 or left uninfected and passaged three times to insure complete infection of all cells in the RAV-6 cultures. Sixty-millimeter petri dishes were seeded with 1 x lo6 cells in 4 ml of GM + Polybrene, infected with ca. 2 x lo4 FFU of RSV, and incubated at 34 or 41”. Duplicate dishes of each virus were incubated at each temperature. The cultures were media changed at 24 hr postinfection with 4 ml of GM + DMSO. Dishes at 35” were media changed at room temperature using media warmed to 35”; 41” dishes were media changed on a slide-warmer set to a surface temperature of 47” using media warmed to 45”. At 96 hr postinfection, the dishes were chilled to 4” for ca. 15 min and then harvested at room temperature and the media stored at -80” for subsequent assay. The cells were discarded. Yields of RSV were determined in the focus assay at 35”. b Harvests of RSV from C/E chick embryo fibroblasts were titered in the focus assay on C/E, C/BE, and C/ C chick embryo fibroblasts. Results are expressed as FFU per milliliter. c Harvests of RSV from C/E chick embryo fibroblasts which had been preinfected with RAV-6 avian leukosis virus were titered in the focus assay on C/E, C/BE, and C/C chick embryo tibroblasts. Results are expressed as FFU per milliliter.

focus formation at 41” on cells preinfected with avian leukosis virus (Linial and Mason, 1973). We favor the explanation that phenotypic leakiness at 41” allows a small fraction of input tsLA337-C to infect the RAV-g-containing cells. In uiuo complementation as well as recombination to wild type between tsL4337-C and RAV-6 might then produce the high yields of virus observed at 41” by allowing tsLA337-C infection to spread in the culture. The results presented in the following sections show that most if not all virus proteins are produced by tsPH734-C at the nonpermissive temperature and become incorporated into defective virus particles but that the two envelope glycoproteins GP85 and GP35 seem to be incorporated

into defective virions at reduced efficiency. TsPH734PR-C Produces Particles at 41’

Defective

Virus

In order to determine whether or not tsPH734-C produces noninfectious virus particles at 41”, infected cells were grown in the presence of i3Hluridine which is incorporated into viral RNA. The material released into the tissue culture fluids was banded to equilibrium on l&50% (w/v) sucrose gradients. wtPR-C or tsPH734-C at either temperature produced RNA-containing particles which banded at a density of 1.15 to 1.16 g/cm”. To verify the identification of these particles as virus, the particles were digested with sodium dodecyl sulfate (SDS) and Pronase and the

448

MASON

AND YEATER

60 are, in order of increasing electrophoretie mobility, P27, P19, and P15 plus P12. PlO is presumably migrating in the P15 plus P12 peak. The peak fraction of GP35, labeled with [3H]fucose, migrated one fraction behind the peak fraction of P27. A parallel analysis of the glycoproteins and proteins of tsPH734-C virus particles (Figs. 1C and D) reveals differences in the glycoprotein pattern between virus radioactively labeled at 35 and 41” as well as between tsPH734-C labeled at either temperature and wtPR-C. The glycoprotein peaks of 13H]fucose-labeled GP85 and GP35 are conspicuously absent from tsPH734-C virions produced at 41”. Peaks of [3H]fucose-labeled macromolecules corresponding to GP85 and GP35 are present in tsPH734-C virions released at 35” but are more heterogeneous in electrophoretic mobility than those of wtPR-C, suggesting that maturation of these glycoproteins may be partially defective at 35”. This suggestion is consistent with the observation that wtPR-C- and tsPH734-C-infected cells

RNA analyzed by velocity sedimentation on sucrose density gradients. All preparations contained a similar high molecular weight 70 S RNA characteristic of avian tumor viruses (data not shown). In the next section, we present an analysis of the proteins and glycoproteins of the defective particles produced by tsPH734-C at 41”. Since GP85 and GP35 are glycoproteins, specific labeling is obtained when cells are grown in the presence of a precursor to the carbohydrate side chains, for example, 13Hlglucosamine or [3Hlfucose. Proteins and Glycoproteins of wtPR-C and tsPH734-C Virus Particles

The results of electrophoresis of viruses labeled with a mixture of [3H]fucose and 14C-labeled amino acids are shown in Fig. 1. Essentially identical protein and glycoprotein profiles are ,obtained with wildtype virus radioactively labeled at either 35 or 41” (Figs. 1A and B). The GP85 peak is indicated. The major internal virus proteins migrating between fractions 40 and I *A

tA

P27

$1

1

1 -I30

I

t f

.___

ts-350

, P.27 !

i30

\-

FRACTION

NUMBER

FIG. 1. Polyacrylamide-gel electrophoresis of wtPR-C and tsPH734-C virions labeled with 13Hlfucose + “C-labeled amino acids. WtPR-C- and tsPH734-C-infected cells were seeded at 3 x 106/100-mm petri dish and incubated at 35 or 41”, with media changing every 24 hr as described in footnote a to Table 2. At 72 hr, the dishes were media changed with 5 ml of 199 labeling medium containing 2% amino acids, 1% DMSO, 50 &i of ‘Glabeled amino acids, and 400 &i of [3Hlfucose. After 14 hr the dishes were placed on ice, the media harvested, and the virus purified as described in Materials and Methods. The virus was collected, and the viral proteins were subjected to electrophoresis on 10% polyacrylamide gels as described in Materials and Methods. The cel!s were washed and stored at -80” for analysis by immune precipitation as described in Table 4. Electrophoresis was from 1eR to right.

HOST

RANGE

MUTANT

give approximately equal yields of 14C-labeled amino acid-labeled virus at 35” (Table 31, but wtPR-C consistently produced 4 to 10 times higher yields of focus-forming virus than tsPH734-C at 35” Since fucose is a terminal sugar of the carbohydrate side chains of glycoproteins, while glucosamine can be incorporated at internal positions, it was possible that the inability to detect [3Hlfucose-labeled GP85 on tsPH734-C virus particles at 41” was due to a defect in completion of carbohydrate side chains. However, similar profiles of glycoprotein were obtained when virus was grown in the presence of a mixture of 13Hlglucosamine and 14C-labeled amino acids (data not shown). Increased TABLE INFECTIVITY

AND

GP85

Virus (1)

CONTENT

449

RSV

heterogeneity was again found in the GP85 and GP35 peaks. Because of the greater amount of glucosamine than fucase in the carbohydrate side chains (Krantz et al., 19761, the glycoproteins were more highly radioactive, and a small peak of GP85 was observed in tsPH734-C virus particles synthesized at 41”. The results of the experiment illustrated in Fig. 1 together with the experiment described above and a third experiment in which virus was labeled with a mixture of 13Hlglucosamine and [14C]serine are summarized in Table 3. The ratios of radioactive sugar in GP85 to radioactive amino acid in P27 or to radioactive amino acid in the total internal proteins of the virus par3

OF VIRIONS:

Replicationb (41”/35”) (3)

OF

wtPR-C Yield

AND tsPH734-C” Relative labeled

of labeled virus’ (4)

(gy! (5) Experiment wtPR-C

35 41 35 41

tsPH734-C

e (6)

1.5 0.053

- 1 (3.2) 3.2 1.14 (1.48) 1.69

0.89 1.22 1.1 0.22

0.36 0.44 0.39 0.073

- l(2.1) 2.1 0.94 (0.87) 0.82

6.2 6.5 5.4 0.7

1.92 1.82 1.57 0.2

f

6.2 3.9 4.3 0.88

1.65 0.9$ 1.03 0.19

2 (glucosamine)

tsPH734-C

Experiment wtPR-C

(gy

1 (fucose)

tsPH734-C

Experiment rutPR-C

amount of 3HGP85 per virion

35 41 35 41

2.4

35 41 35 41

0.5

0.073

3 (glucosamine)

0.028

1 (0.87) 0.87 0.?7 (0.44) 0.38

a The data from Fig. 1 (Experiment 1) are summarized, together with an expe+ment in which virus was labeled with a mixture of [3Hlglucosamine (80 &i/ml) + ‘*C-labeled amino acids (10 p&/ml) (Experiment 2) and an experiment in which virus was labeled with a mixture of [3H]glucosamine (86 @X/ml) + [‘Wserine (12.5 &i/ml). Labeling cohditions remained essentially as described in the legend to Fig. 1: ’ FFU per milliliter were determined on aliquots of the radioactive virus obtained prior to purification and the ratios of FFU per milliliter at 41 to 35” are presented. ’ Relative yields of “C-labeled amino acid label in virus were measured by summing the peak fractions on the virus gradients. The yield of “C-labeled amino acid in wtPR-C is taken as unity in each experiment. The 41 to 35” ratios are given in parentheses. d Ratio of [3H]glucosamine or [3H]fucose label in GP85 region to W-labeled amino acid label in the P27 region of the polyacrylamide gels. e Ratio of [3Hlglucosamine or [3H]fucose in GP85 region to W-labeled amino acid label in the internal core protein region (approximately fractions 40-60).

450

MASON

AND

titles plus PlO are given in columns 5 and 6 of Table 3. In each experiment, a relative reduction in the radioactive GP85 per virion is observed when tsPH734-C is grown at 41”, the greatest reduction being ca. eightfold (Table 3, Experiment 2). Since the relative amount of radioactive GP85 in tsPH734-C virus particles synthesized at 41” is probably overestimated for Experiments 2 and 3 (Table 3) by a factor of ca. 2 because of a high background of radioactive carbohydrate throughout the gels, which was not accounted for in making the calculations, the radioactive GP85 to P27 or GP85 to internal protein ratio is probably reduced 7- to lo-fold when tsPH734 virus particles are synthesized at 41”. Calculations of the GP35 content of virions have not been made because of the relatively low number of counts per minute of radioactive sugar in GP35 with respect to the background of carbohydrate across the gels, as was mentioned above. It is clear from Fig. 1, however, that a substantial reduction in the radioactive GP35 content of virions has occurred. A similar conclusion was obtained following 13Hlglucosamine labeling. Consideration of the radioactive amino acid-labeled material corn&rating with radioactive fucose-labeled GP85 (Fig. 1) reveals that a substantial amount of amino acid-labeled material remained when tsPH734-C virions were grown at 41”. One possible explanation is that the peak of amino acid label at the position of GP85 occurs against a background of amino acid label throughout the gel. Sample calculations indicate, however, that this explanation is probably not adequate to account for the apparently disproportionate amount of amino acid label in this region of the gels of tsPH734-C at 41” (Fig. 1D). Two other explanations remain: Either a reduction has occurred in the specific activity of carbohydrate precursor incorporated into GP85 synthesized by tsPH734-C at 41”, or a nonglycosylated protein coelectrophoreses with GP85. These alternatives have not yet been resolved. If tsPH734-C is defective in the incorporation of GP85 and GP35 into virus particles at 41”, we might expect to find an

YEATER

intracellular accumulation of these glycoproteins at 41”. Data are now presented which indicate an accumulation of [3H]glucosamine-labeled GP85 at 35 or 41” in tsPH734-C-infected cells in excess of the amounts found in cells infected with z.utPR-C . GP85 in wtPR-C- and tsPH734-C-Infected Cells

Figure 2 shows the analysis by electrophoresis on polyacrylamide gels of immune precipitates obtained with antiGP85 serum from cells labeled with 13Hlglucosamine. The results of nonspecific precipitation from each lysate when normal rabbit serum substitutes for specific serum are also shown. It is apparent from the results of precipitation with specific serum that a significantly greater percentage of a radioactive glycoprotein with the electrophoretic mobility of GP85 is found in lysates of tsPH734-C-infected cells at both permissive and nonpermissive temperatures. GP35 or a molecule with similar electrophoretic mobility also appears, perhaps due to self-aggregation or disulfide bond formation with GP85 (Leamnson and Halpern, 1976). However, some or all of this GP35-like material may be cellular since a similar peak is found in uninfected cell lysates (Halpern et al., 1975). The results obtained by immune precipitation of GP85 are summarized in Table 4 (Experiment 2). The percentage of cell-associated [3H]glucosamine counts per minute in GP85 in tsPH734-C-infected cells at each temperature is seen to be twoto threefold higher than in wtPR-C-infected cells at the same temperature. The accumulation in tsPH734-C cells at the permissive temperature could reflect a partial defect in GP85 maturation even at 35”, leading to accumulation of aberrant by-products or to a slower rate of maturation and release. The ratio of cell-associated to viral GP85 t3H counts per minute) in tsPH734-C-infected cultures was ca. 12 at 35” and ca. 100 at 41”. For wtPR-Cinfected cultures, the ratio was ca. l-2 at either temperature. In contrast to the results obtained by glucosamine labeling, the percentage of

HOST RANGE

G 24kR

MUTANT

GP85

IO

20

451

OF RSV

tF

30

40

50

FRACTION

480

60

10

20

30

40

50

NUMBER

FIG. 2. Analysis of anti-GP85 immune precipitates from wtPR-C- and tsPH734-C-infected cells. Labeling of the infected cells with [3Hlglucosamine + Y-labeled amino acids was as described in footnote a to Table 3 (Experiment 2). Immune precipitates were prepared from 5 x 10” “H cpm and analyzed by electrophoresis on 10% polyacqdamide gels as described in Material and Methods. Electrophoresis was from left to right. Only the 3H label is shown. (A, B, E, and P), Anti-GP85 precipitates. (C, D, G, and H), Precipitates occurring when normal rabbit serum substitutes for anti-GP85 serum.

13H]fucose label in cell-associated GP85 in tsPH734-C-infected cells at 35 and 41” is the same as in wtPR-C-infected cells at the corresponding temperatures (Table 4, Experiment 1). Since fucose has been shown to be added at or near the plasma membrane in other systems (Parkhouse, 1973), these results may imply that GP85 maturation is partially blocked prior to incorporation into the plasma membrane’ of tsPH734-C-infected cells. In order to test the possibility that the ratio of GP85 to virus internal proteins found in virions reflects the ratio associated with cells, the amount of virus antigen in cells detectable in the COFAL assay (Heubner et al., 1964) was measured, as described in Materials and Methods. wtPR-C- and tsPH734-C-infected cells contained similar amounts of virus antigen detectable in this assay. This result is consistent, but not definitive, evidence that

the group-specific virus internal proteins (Duesberg et al., 1968) are produced in equivalent amounts under all conditions. This result is also consistent with the hypothesis that the mutation in tsPH734-C specifically affects GP85 and GP35. In the next section, we present evidence that tsPH734-C is not complemented for replication at 41” on fibroblasts preinfected with the Bryan strain of Rous sarcoma virus, a noninfectious variant of Rous sarcoma virus which produces virions lacking detectable GP85 and perhaps GP35 as well (Scheele and Hanafusa, 1971). Lack

of in Vivo Complementation tsPH734-C by BH-RSV( -)

of

The results of replication of wtPR-C, tsPH734-C, tsLA337-C, and tsLA338-C on fibroblasts preinfected with BH-RSV(--1 are shown in Table 5. Only tsPH734-C failed to be complemented to a significant

452

MASON INTRACELLULAR

Virus (1) Experiment wtPR-C

Temperature

tsPH734-C Uninfectedd

TABLE 4 GP85 IN wtPR-C- AND tsPH734-C-hmzTm CELLS~ Radioactive precursor Total TCA-preCounts in cipitable counts* GP85’ (3) (4) (it;

35 41 35 41

[3H]fucose F3Hlfucose [3Hlfucose 13HIfucose

1 1.9 2.4 1.7

x x x x

106 106 lo6 106

1.43 2.8 0.91 1.87

35 41 35 41

[3Hlglucosamine [3Hlglucosamine [3Hlglucosamine [3H]glucosamine

1.3 1.3 1.5 0.45

x x x x

10’ 10’ 10’ 10’

1.49 4.0 6.5 11.6

35 41 35 41 41

[3Hlglucosamine [3Hlglucosamine [3Hlglucosamine [3H]glucosamine [3Hlglucosamine

1.4 0.43 1.5 0.8

x x x x 2.5 x

107 10’ 10’ IO’ lo6

0.69 0.98 2.2 3.9 0.29

2

tsPH734-C Experiment wtPR-C

OF

1

tsPH734-C Experiment wtPR-C

LEVELS

AND YEATER

3

CI

o Summary of anti-GP85 immune precipitations from cells of experiments described in Table 3. * Total TCA-precipitable 3H counts per minute in clariiied cell lysates. ’ Ratio of 3H counts per minute in GP85 region of 10% polyacrylamide gels to TCA-precipitable 3H counts per minute in clarified cell lysates. Correction was not made for differences in counting efficiency between TCA precipitation and polyacrylamide-gel fractions. Correction was made for 3H counts per minute in the GP85 region of the gels detected when normal rabbit serum substituted for anti-GP85 serum. ’ Analysis of anti-GP85:immune precipitate of uninfected C/B chick embryo fibroblasts used in Experiment 3. The high background in the GP85 region may have reflected the fact that these cells ‘showed a low level of &f-expression at 41”.

extent for replication at 41” on BHRSV(-)-infected cells. The defectiveness of these BH-RSV(-)-infected cells was * therefore examined. An analysis by gel electrophor%s of the virions released by BH-RSV(-)-infected cells or by the same ceIIs follbwing superinfection with the transformation de&+tive virus, tcZPR-C, gave essentially the results described by Scheeie and danafusa (1971). BH-RSV( -> virus were def;cient ‘in GP85 and GP35. The ratio of’ [$I]glucosamine counts per minute in the t&%5 region of the gel to i4C-labeled an&o acid counts per minute in the core protein, P27, was distinctly. lower ‘for BH-RSV( -) virus particles, as compared to the infectious BH-RSV( tdPR-C) v+us particles,. While defective BH-RSV( -)virus, particles appear similar ta noninfecitious tsPH734-C virus particles produced under

nonpermissive conditions, antiLGP85 immpne prec
HOST TABLE BH-RSV( Virus (1)

-)

DOES NOT Replication temperature (“Cl (2)

wtPR-C

tsPH734-C clone 1 tsPH734-C clone 3 tsLA337-C

tsLA338-C

35 41 41135 35 41 41135 35 41 41135 35 41 41/35 35 41 41/35

RANGE

MUTANT

5

COMPLEMENT tsPH734-C” Replication on BHRSV( -)-infected C/B cells, focus assays* C/BE (3) 5.3 x 104 7.8 x lo4 1.5 9.4 x 105 2.5 x lo4 0.027 1.4 x 104 3.8 x 10% 0.027 4.3 x 104 5.2 x lo3 0.12 1.3 x 105 9 x 10’ 0.69

c/c (4) ca. 10’ 0 ca. 4 x 10’ 0 ca. 10’ 0 Not

tested

Not

tested

due to overlapping defects in virus envelope glycoprotein common to both BHRSV( - ) and tsPH734-C . is Replication

453

RSV

have examined the glycoprotein of virions of tdtsPH734-C, a variant of tsPH734-C unable to transform flbroblasts at 35 or 41”. TdtsPH734-C was as defective as tsPH734C in the incorporation of GP85 and GP35 into virus particles synthesized at 41” (data not shown). To determine whether tdtsPH734-C is defective in virus replication at 41”, aliquots were added to cultures of BHRSV(-j-infected cells at 35 or 41”, and the helper activity of tdtsPH734-C for production of infectious virus was measured. tdtsPH734-C was defective in the ability to serve as a helper for the noninfectious BHRSV(-) at 41” but not at 35” (data not shown). We conclude therefore that tdtsPH734-C has retained the mutation of tsPH734-C for replication at 41” DISCUSSION

0 BH-RSV(-)-infected C/B chick embryo tibroblasts were seeded at 1 x 106/60-mm petri dish in GM + Polybrene, superinfected at a multiplicity of infection of ca. 0.02 with the indicated virus, and incubated at 35 or 41”. Duplicates were done at each temperature. The cultures were media changed at 28 hr and the virus-containing media harvested at 72 hr as described in footnote a to Table 2. b Harvests of RSV were titered in the focus assays on C/BE and on C/C chick embryo libroblasts. Results are expressed as FFU per milliliter. The low efficiency of focus formation of the harvests on C/C cells indicates that the subgroup C host range specificity of the superinfecting virus has been maintained. Although both PR-C- and BH-RSV-type foci seemed to be present in the focus assays on C/BE cells in roughly equivalent amounts, no attempt was made to quantitate each type; thus, only total FFU per milliliter were recorded.

TdtsPH734-C

OF

Defective

at

41” Lai and Duesberg (1972) have shown that the average size of the glycopeptides derived by proteolytic cleavage of virion glycoproteins is influenced by the transformed state of the infected cell. To determine whether detection of GP85 or GP35 by glucosamine or fucose labeling is affected by the transformed state of the cell, we

GP85 and GP35 are detected as a disulfide-linked dimer in infected cells as well as in the virus released by these cells (Leamnson and Halpern, 1976); moreover, J. England et al. (personal communication) have obtained indirect evidence consistent with the possibility that GP85 and GP35 initially arise by proteolytic cleavage of a high molecular weight precursor protein. In the present publication, we describe a temperature-sensitive mutant of the Prague strain of Rous sarcoma virus, tsPH734PR-C, which is defective in the specification of virus host range and in the incorporation of both GP85 and GP35 into virus particles at 41”. In view of the findings described above, it seems possible that a defect in either GP85 or GP35 might prevent incorporation of both glycoproteins into virus. These considerations indicate the difficulty in determining, from the available data, whether tsPH734PR-C has a defect in GP85, GP35, or both. Several aspects of the temperature-sensitive replication mutant, tsPH734PR-C, require immediate investigation. First, direct proof of an alteration in the primary structure of GP85 and/or GP35 must be obtained in order to provide support for our hypothesis that there is a mutation in the virus gene(s) which codes for these pro-

MASON AND YEATER

454 ,. 1 A

R

(-I- anti

1

(GP85)

B

GPBJ t

6

c

D

GP85 t

6

PR-Cl-anti

(GP85)j

2p

GP85

R(-)-NRS

10

R (td

R(sPR-C)-NRS

dye

GP85

+

1

6 E

10

20

30

40

50

60

70

FRACTION

10

20

30

40

50

60

70

NUMBER

FIG. 3. Analysis of anti-GP85-immune precipitates from BH-RSV(-)and BH-RSV(tdPR-C)-infected cells. BH-RSV(-)-infected C/B chick embryo tibroblasts (described in Table 5) either not superinfected or previously superinfected with tdPR-C were seeded at 2.5 x 106/100-mm petri dish in GM + Polybrene and incubated at 35 or 4I”, with media changing two times daily as described in the legend to Fig. 1. At 56 hr, the dishes were media changed with 5 ml of 199 labeling medium containing 2% amino acids, 1% DMSO, 50 $i of 14C-labeled amino acids, and 400 &i of [3H]glucosamine. After 15 hr, the virus was purified and analyxed by gel electrophoresis, with the results described in the text. The cells were washed and stored at -80” for subsequent immune precipitation. Immune precipitates were prepared from 1 x lo6 3H cpm of cells labeled at 41” and analyzed by electrophoresis on 10% polyacrylamide gels. Only the 3H counts per minute are shown. Electrophoresis was from left to right. (A), BH-RSV(-)-anti-GP85. (B), BH-RSV(tdPR-C)-antiGP85. (C), BH-RSV(-)-normal rabbit serum (NRS). (D), BH-RSV(tdPR-C)-normal rabbit serum (NRS). TABLE INTRACELLULAR

Virus (1)

6

LEVELS OF GP85 IN INFECTED CELLS”

Temperature ‘;zc,’

BH-RSV(-)-

Total incorporation of [3H]glucosamine* (cpm) (3)

3H counts Gg5’ 2;

BH-RSV(-) 41 2.4 x lo6 5 0.12 BH-RSV(tdPR-C) 41 3.8 x lo6 2.1 a Summary of anti-GP85-immune precipitations from cells of experiment described in Fig. 3. b Total TCA-precipitable 3H counts per minute in clarified cell lysates. c See footnote c. Table 4.

teins. Second, since tsPH734PR-C is unable to synthesize infectious virus of subgroup C at the nonpermissive temperature (41”), the mutation of tsPH734PR-C should be mapped on the virus genome relative to

the genetic marker(s) for virus host range.. This goal might be achieved through analysis of the primary structure of the virus RNA genomes of genetic recombinant virus isolated following recombination between the host range and temperaturesensitive genetic markers (Joho et al., 1976; Duesberg et aZ., 1976). Finally, the accumulation of “excess” GP85 in tsPH734PR-C-infected cells should be investigated in greater detail. Resolutions of these problems may provide information on the site or sites on the primary structure of the viral glycoproteins responsible for specifying virus host range, as well as information on the process of virus glycoprotein maturation and insertion into the virus envelope. ACKNOWLEDGMENTS We are especially indebted to Dr. Dani Bolognesi (Duke University Medical Center, Durham, North

HOST

RANGE

Carolina) for the gift of anti-GP85 serum, to Drs. Young Chen and Peter Vogt (University of Southern California School of Medicine, Los Angeles, California) for the BH-RSV(-)-infected cells, to Dr. Lyman Crittendon (Regional Poultry Research Laboratory, East Lansing, Michigan) for embryonated eggs of line 15 chickens, and to Dr. Maxine Linial (Fred Hutchinson Cancer Center, Seattle, Washington) for tcZPR-C. To Drs. Michael Halpern, Robert Leamnson, John Taylor, and Jesse Summers we are grateful for many helpful suggestions. We acknowledge the assistance in the laboratory of Charles Keegan, Melva Petersen, and Ellis Rucker. This research was supported by USPHS Grants CA16641, CA-06927, and RR-05539 and by an appropriation from the Commonwealth of Pennsylvania. REFERENCES BONAR, R. A., ISHIZAKI, R., and BEARD, J. W. (1972). Immunoelectrophoretic analysis of avian ribonucleic acid tumor virus group-specific antigens. J. Virol. 9, 90-95. DUESBERG, P. H., ROBINSON, H. L., ROBINSON, W. S., HUEBNER, R. J., and TURNER, H. C. (1968). Proteins of Rous sarcoma virus. Virology 36, 7386. DUESBERG, P. H., and VOGT, P. K. (1973). RNA species obtained from clonal lines of avian sarcoma and from avian leukosis virus. Virology 54, 207-219. DUESBERG, P. H., WANG, L.-H., MELLON, P., MASON, W. S., and VOGT, P. K. (1976). In “Proceedings” of the ICN-UCLA Symposium on Animal Virology” (D. Baltimore, A. Huang, and C. F. Fox, eds.). Vol. 4, pp. 107-125. Academic Press, New York. FRIIS, R. R., and HUNTER, E. (1973). A temperaturesensitive mutant of Rous sarcoma virus that is defective for replication. Virology 53, 479-483. FRIIS, R. R., MASON, W. S., CHEN, Y. C., and HALPERN, M. S. (1975). A replication defective mutant of Rous sarcoma virus which fails to make a functional reverse transcriptase. Virology 64.49-62. HALPERN, M. S., BOLOGNESI, D. P. and LEWANDOWSKI, L. J. (1974). Isolation of the major ‘viral glycoprotein and a putative precursor from cells transformed by avian sarcoma viruses. Proc. Nut. Acad. Sci. USA 71, 2342-2346. HALPERN, M. S., BOLOGNESI, D. P., FRIIS, R. R., and MASON, W. S. (1975). Expression of the major viral glycoprotein of avian tumor virus in cells of chf(+) chick embryos. J. Viral. 15, 1131-1140. HALPERN, M. S., BOL~CNESI, D. P., and FRIIS, R. R. (19761. Viral glycoprotein synthesis studies in an established line of Japanese quail embryo cells infected with the Bryan high-titer strain of Rous sarcoma virus. J. Virol. 18, 504-510. HEUBNER, R. F., ARMSTRONG, D., OKUYAN, M.,

MUTANT

OF

RSV

455

SARMA, P. S., and TURNER, H. C. (1964). Specific complement-fixing viral antigens in hamster and guinea pig tumors induced by the Schmidt-Ruppin strain of avian sarcoma. Proc. Nat. Acad. Sci. USA 51, 742-750. HUNTER, E., and VOGT, P. K. (1976). Temperature sensitive mutants of avian sarcoma viruses. Genetic recombination with wild type sarcoma virus and physiological analysis of multiple mutants. Virology 69, 23-34. HUNTER, E., HAYMAN, M. J., RONGEY, R. W., and VOGT, P. K. (1976). An avian sarcoma virus mutant that is temperature sensitive for virion assembly. Virology 69, 35-49. JOHO, R. H., STOLL, E., FRIIS, R. R., BILLETER, M. A., and WEISSMANN, C. (1976). In “Proceedings of the ICN-UCLA Symposium on Animal Virology” (D. Baltimore, A. Huang, and C. F. Fox, eds.). Vol. 4, pp. 127-145. Academic Press, New York. KRANTZ, M. J., LEE, Y. C., and HUNG, P. P. (1976). Characterization and comparison of the major glycoprotein from three strains of Rous sarcoma virus. Arch. Biochem. Biophys. 174, 66-73. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-682. LAI, M. C., and DUESBERG, P. K. (1972). Differences between the envelope glycoproteins and glycopeptides of avian tumor viruses released from transformed and from non-transformed cells. Virology 50, 359-372. LEAMNSON, R. N., and HALPERN, M. S. (1976). Subunit structure of the glycoprotein complex of avian tumor virus. J. Virol. 18, 956-968. LINIAL, M., and MASON, W. S. (1973). Characterization of two conditional early mutants of Rous sarcoma virus. Virology 53, 258-273. MASON, W. S., FRIIS, R. R., LINIAL, M., and VOGT, P. K. (1974). Determination of the defective function in two early mutants of Rous sarcoma virus. Virology 61, 559-574. MORGAN, J. F., MORTON, H. J., and PARKER, R. C. (1950). Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. Proc. Sot. Exp. Biol. Med. 13, 1-8. PARKHOUSE, R. M. E. (1973). Assembly and secretion of immunoglobulin M (IgM) by plasma cells and lymphocytes. Transplant. Rev. 14, 131-144. SARMA, P. S., LOG, T., HUEBNER, R. J., and TURNER, H. C. (1969). Studies of avian leukosis group-specific complement fixing serum antibodies in pigeons. Virology 37, 480-483. SCHEELE, C. M., and HANAFUSA, H. (1971). Proteins of helper-dependent RSV. Virology 45,401-410. SUZAWA, H., SUGIMORI, T., MOIRA, Y., and SHIMIZA, T. (1966). Specific complement test fixation of Rous sarcoma with pigeon serum. Nut. Inst. Anim. Health Q. 6, 208-215.

456

MASONANDYEATER

K., and VOGT, P. K. (1969al. Temperature sensitive mutants of an avian sarcoma virus. Virology 39, 930-931. TOYOSHIMA, K., and VOGT, P. K. (1969b). Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions. Virology 38, 414-426. VOGT, P. K. (1969). Focus assay of Rous sarcoma virus. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.1, pp. 198-211. Academic Press, New York. VOGT, P. K., TOYOSHIMA, K., and YOSHII, S. (1970). Factors promoting avian tumor virus infections. In “Defectivite, Demasquage, et Stimulations des Virus Oncogenes,” International Symposium on Tumor Viruses, 2nd, Royaumont, pp. 229-238. VOGT, P. K., WEISS, R. A., and HANAFUSA, H. (1974). Proposal for numbering mutants of avian TOYOSHIMA,

leukosis and sarcoma viruses. J. Virol. 13, 551554. VOGT, V. M., EISENMANN, R., and DIGGELMANN, H. (1975). Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide. J. Mol. Biol. 96,471-493. WEISS, R. A., MASON, W. S., and VOGT, P. K. (1973). Genetic recombinants and heterozygotes derived from endogenous and exogenous avian RNA tumor viruses. Virology 52, 535-552. WYKE, J. A. (19731. The selective isolation of temperature-sensitive mutants of Rous sarcoma virus. Virology 52, 587-590. WYKE, J. A., and LINIAL, M. (1973). Temperaturesensitive avian sarcoma viruses: A physiological characterization of twenty mutants. Virology 53, 152-161.