The defective maturation of viral progeny with a temperature-sensitive mutant of avian sarcoma virus

73, 259-272 (19761

VIROLOGY

The Defective Maturation of Viral Progeny with a TemperatureSensitive Mutant of Avian Sarcoma Virus ROBERT fiir Virologie,

Institut

Fachbereich

R. FRIIS’

Robert Koch Znstitut,

of Anatomy

HAJIME

Humanmedizin, Justus Liebig Stra/3e 107, West Germany

HANS

The Wistar Institute

AND

Uniuersitiit,

63 Giessen, Frankfurter

GELDERBLOM

1 Berlin

West-65, Nordufer

MICHAEL

S. HALPERN

and Biology,

OGURA

Thirty-sixth 19104

Accepted April

20, West Germany

Street at Spruce, Philadelphia,

Pennsylvania

7, 1976

LA334 (previously ts 75) has been identified as a mutant exhibiting two genetic lesions affecting late functions of the avian sarcoma virus genome. One causes the transformed phenotype to be temperature sensitive, and the other induces a rapidly reversible inhibition of progeny virus production at the nonpermissive temperature. The nature of the latter defect has been investigated in this study with the following findings: (1) no new protein synthesis was needed to initiate synthesis of infectious virus after a shift from the nonpermissive to the permissive temperature; (2) significant amounts of physical particles (20 to 70% of that produced at the permissive temperature) were produced at the nonpermissive temperature; and (3) intracellular accumulation of structures resembling budding virus, approximately 80% of which were aberrant, were observed in mutant-infected cells at the nonpermissive temperature. The possibility that a defect in the synthesis of the viral envelope glycoprotein, gp85, would account for these findings prompted an investigation of the expression of gp85 in LA334-infected cells. Intracellular synthesis of gp85 in normal amounts was shown by immunoprecipitation; however, only inefficient interference against superinfecting virus could be observed with mutant-infected cells at the nonpermissive temperature. Immunoferritin electron microscopic observations also indicated the presence of envelope glycoprotein in association with budding virions of normal morphology, but in contrast, little or no glycoprotein was associated with the budding structures of aberrant morphology. It is concluded that the primary virus defect is not related to expression of gp85; rather, on the basis of the abnormal core assembly, it is proposed that a defect exists in one of the core structural proteins. This hypothesis is discussed in the light of known properties of the mutant and of certain recent observations on the composition of the noninfectious virus produced by infected cells at the nonpermissive temperature. INTRODUCTION

Although LA334 (previously designated ts 75) was the first temperature-sensitive mutant RNA tumor virus to be isolated * Author addressed.

to whom requests for reprints

should be

(Toyoshima and Vogt, 19691, it has proved to be one of the most difficult to analyze with respect to the defective viral function. On the basis of temperature shift experiments undertaken to examine the appearante of infectious viral progeny after shift 259

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

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from the nonpermissive temperature (42”) to the permissive (35”), LA334 was recognized to have a late defect (Friis et al., 1971). This same study concluded that the defect prevented release of viral particles and, on the basis of tests for the frequency of reversion to wild-type with the transformation marker, argued that there was probably a single genetic lesion in LA334. The existence of two independent genetic lesions was, however, conclusively shown by Owada and Toyoshima (1973), who were able to isolate recombinants of LA334 with avian leukosis viruses that had become wild-type with respect to replication of viral progeny but had remained temperature sensitive for transformation. Additional work with LA334 has been directed mainly to analyzing the nature of the defect in replication of virus progeny. Katz and Vogt (1971) used various metabolic inhibitors to investigate the effect on temperature shift and concluded that production of infectious progeny after shift to the permissive temperature was dependent on new protein synthesis, but not on new RNA or DNA synthesis. Furthermore, on the basis of experiments utilizing radioisotopic pulse labeling of infected cells, these authors suggested that newly released infectious virus obtained after shift to the permissive temperature was enriched for two labeled proteins: a large one thought to be the major viral envelope glycoprotein, gp85, and a smaller one, probably ~10, ~12, or ~15. This communication is concerned with the assembly of the viral structural components in cells infected with LA334 at the nonpermissive temperature. On the basis of the evidence presented, some of the previous information about the nature of the defective replicative function mut be reinterpreted. A new hypothesis is suggested which, in addition to explaining what we know of the biological and biochemical properties of LA334 replication, may suggest useful experiments for obtaining general information about the morphogenesis of the RNA tumor viruses. MATERIALS

AND

METHODS

Cells and viruses. Fertile White leghorn eggs of the C/O phenotype were gener-

ET AL.

ously supplied by Dr. E. Vielitz, Lohmann Tierzucht, Cuxhaven, West Germany. White Leghorn eggs of the C/BDE phenotype were obtained through the courtesy of Dr. W. S. Mason, The Institute for Cancer Research, Philadelphia, Pa., and from Heisdorf and Nelson Farms, Redmond, Wash. Embryos from these eggs were used in the preparation of chick embryo cell (CEC) cultures that were tested for the expression of the chick helper factor (chf) (Hanafusa et al., 1970) according to two methods previously described (Friis et al., 1975). Only CEC conclusively shown to be chf negative were used in these investigations. A stock of LA334 (Toyoshima and Vogt, 1969), a mutant obtained from 5-azacytidine mutagenized avian sarcoma virus B77, subgroup C, was prepared as previously described (Friis et al., 1971). The nomenclature for mutants follows the suggestion of Vogt et al. (1974). Mutants used in this study for control purposes include: LA338, a 5-azacytidine-induced mutant of Prague strain Rous sarcoma virus, subgroup C, that is temperature sensitive for both replication and transformation (Wyke and Linial, 1973); LA336, a 5-azacytidine-induced mutant of the B77 strain of Rous sarcoma virus, subgroup C, that is temperature sensitive for both replication and transformation; and LA672, a 5-azacytidine-induced mutant of Prague strain Rous sarcoma virus, subgroup A, that is defective only for viral replication (Friis and Hunter, 1973; Friis et al., 1975). The following avian leukosis viruses, Rous-associated viruses (RAV), were used in these experiments: RAV-3, subgroup A, and RAV-49, subgroup C. The wild-type parent ofLA334, B77 wt, subgroup C, was also used for many control experiments. Media. Eagle’s Dulbecco-modified medium was purchased as a powder from Flow Laboratories (Bonn, West Germany) and was used to prepare both growth and agar overlay media. Media were supplemented with 10% tryptose phosphate broth, 5% heat-inactivated calf serum, and antibiotics. For overlays used in the focus test, 0.9% Bacto Difco agar (Difco Corp., Ann Arbor, Mich. ) was included in the medium. For preparation of stocks, radio-

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active-labeling procedures, and other experiments involving maintenance of confluently transformed cultures, 1% dimethyl sulfoxicle was included (Vogt et al., 1970). Focus tests were performed essentially as described by Rubin (19601, except that at the time of infection, polybrene (Aldrich Chemical Co., Milwaukee, Wis. 1 was included in the medium at a concentration of 2 pg/ml to enhance virus adsorption (Vogt et al., 1970). Electron microscopy and immunoelectron microscopy. In order to stuay virus expression and the morphology of LA334 infected CEC, confluent cultures were fixed rapidly in situ by adding 2.5% cold glutaraldehycle to the monolayers. The cells were then scraped from the plastic dishes with a rubber policeman, postfixed in 1% OsO1, and after agar inclusion processed in a conventional manner for Epon embedding (Gelderblom et al., 1974). Immunological analysis of viral envelope antigens was performed using chicken immune sera in an indirect hybrid antibody technique (HSimmerling et al., 1968). Rabbit hybrid antibody with immunological specificities against chicken IgG and horse spleen ferritin was prepared as described previously (Gelderblom et al., 1972). LA334-infected CEC were prefixed for 5 min with 0.5% glutaraldehycle, washed three times with phosphate buffered saline, and incubated with different types of chicken antisera, exhibiting specificity for either subgroup C or subgroup A of the avian sarcoma-leukosis complex, each at a dilution of l:lO, for 20 min at 25”. After reaction, the cultures were washed three times and the hybrid antibody was added to a concentration of 0.5 mg/ml for a 20-min incubation at 25”. Finally, after further washing, 1 mg/ml of ferritin was applied. After five further washings, the samples were fixed with 2.5% glutaralclehyde and processed in situ for Epon embedding. Ultrathin sections were prepared and were stained with alkaline lead citrate (Venable and Coggeshall, 1965). Radioisotopic labeling of viral proteins. A pulse-chase experiment was performed using [35S]methionine (300 Ci/mmol; Amersham Buchler Corp., Braunschweig,

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West Germany) in methionine-free Eagle’s minimal essential medium supplemented with 5% calf serum, 1% DMSO, and antibiotics. The isotope was used at 100 &i/ml for an interval of 15 min, followed by two washes in growth medium, and incubation for 165 min in Eagle’s minimal essential medium supplemented with 120 mg/liter of cold methionine. The effect of cycloheximicle (Sigma Chemical Co., St. Louis, MO. 1 on the release of radioactively labeled viral particles in this pulse-chase experiment was monitored by determining the amount of radioactivity from each culture supernatant that appeared in an equilibrium sucrose density gradient band between 1.15 and 1.16 g/cm3. To label viral structural proteins, a “Hamino acid mixture (New England Nuclear Corp., Boston, Mass.) was included in an amino acid-free Eagle’s medium supplemented with 5% calf serum, 1% DMSO, and antibiotics, at a final concentration of 300 &i/ml. Labeling was performed for 10 hr, after which the supernatants were prepared for eventual purification of radioactively labeled viral particles, and the cells lysed for immunoprecipitation experiments as described below. For the specific recognition of glycoproteins, cells were also labeled with 3H-fucose (15 Ci/mmol; New England Nuclear Corp.) in Eagle’s medium containing only l/4 of the normal glucose concentration, but supplemented with 5% calf serum, 1% DMSO, and antibiotics. The 3H-fucose was also used in these experiments at a final concentration of 300 j.Ki/ml. Immune precipitation and analysis of intracellular gp85 using SDS polyacrylamide gel electrophoresis. The use of the nonionic detergent nonidet P-40 for the preparation of intracellular lysates, as well as the method of indirect immune precipitation with a monospecific antiserum to the purified gp85 of the subgroup C strain of Prague Rous sarcoma virus (Pr0, have been described (Halpern et al., 1974). The anti-gp85 serum was generously provided by Dr. Dani Bolognesi, Duke University, Durham, N.C. Immune precipitates were prepared for electrophoresis on 5% SDS polyacrylamide gels and radioactivity determined also as described

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ET AL.

(Halpern et al., 1974). 14C-Amino acid-labeled B77 protein was coelectrophoresed on each gel to serve as positional marker. RESULTS

Reversibility of the Replication Block without New Protein Synthesis after Temperature Shift A shift from the nonpermissive (42”) to the permissive temperature (35”) with LA334-infected CEC is accompanied by a sharp rise in titer of infectious virus within 1 hr. One reason for the rapid release of infectious virus might be that nonfunctional viral protein synthesized in LA334-infected cells at the nonpermissive temperature could be converted to a functional form after shift, thus allowing an early start in the assembly of infectious virus, prior to the synthesis of new protein. This hypothesis was considered and discarded by Katz and Vogt (1971) as a result of experiments in which the inhibitor of protein synthesis, cycloheximide, appeared to completely suppress the production of infectious progeny after temperature shift. We have performed further experiments of this type because the discovery of precursors for the viral internal proteins (~27, p19, ~15, and ~12) (Vogt and Eisenmann, 1973; Vogt et al., 1975) and envelope glycoprotein (gp85) (Halpern et al., 19741, suggested that pools of precursors might exist at the nonpermissive temperature whose correct processing to virion structural proteins might take place soon after shift to the permissive temperature. Since pools of precursors might be expected to have a limited half-life, preliminary experiments were required to determine the minimum time during which cycloheximide might be able to establish significant inhibition of viral protein synthesis in LA334-infected cells. Inhibition in conjunction with a temperature shift should then permit an assessment to be made as to whether new protein synthesis is needed for the release of infectious virus in the first hour after shift of the cells to 35”. Table 1 shows the results of the preliminary experiments which were performed with B77 wt-infected cells. A 30min treatment with cycloheximide beginning just 5 min before a 15-min pulse label

TABLE

1

CONDITIONS FOR A CYCLOHEXIMIDE BLOCK EXPERIMENT Sam- Time of cycle- Time of Counts per Percentheximide ?3Imethiomin age of conele treatment? nine pulsed [Wlmethio trol re(min) (min) nine in re- j/ leased vileased virus rw 1 2 3 4 5 6

None +15 to +45 +5 to +35 Oto+30 -5 to t25 -15 to +15

0 to +15 0 to +15 0 to +15 Oto+15 0 to +15 0 to +15

1.2 x 7.1 x 9.0 x 2.5x <1 x
10” lo” 10” 10’ 104 10’ I

100 59 7.5 2.1 <1
N CEC infected with B77 wt were investigated for the inhibitory effect of a 30-min treatment with cycloheximide (5 pgiml) on the release of radioactively labeled virus at 42”. Zero (0) minute was defined as the time when the radioactive pulse was initiated; times indicated as negative were before the pulse b The pulse was performed for 15 min using l%Imethionine at a final activity of 100 pCi/ml as described above in Materials and Methods. The chase was for 165 min after which the supernatants were harvested. c Virus-specific radioactivity was determined after purification of virus by sedimentation through a 20 to 50% sucrose gradient to equilibrium according to methods recently described (Friis et al _, 1975). The virus specific radioactivity was recovered in a band at a density of 1.15 to 1.16 g/cm:‘.

with [35S]methionine was sufficient to reduce the yield of radioactivity released during the following 3 hr as virus, by more than 99%. Inhibition of total cellular protein synthesis was reduced by 95% within 5 min after cycloheximide treatment. On the assumption that synthesis of all viral protein is inhibited with the same kinetics by treatment with 5 Fglml of cycloheximide, a shift experiment was then performed, and the results are presented in Table 2. Cycloheximide was added at various time before the shift. It was found that even when cycloheximide treatment was begun 30 min before the shift, the titer of infectious progeny rose more than lofold during the first hour after shift. When cycloheximide inhibition was applied shortly before shift (5 or 15 min), titers rose 50-fold within 1 hr after shift. The results show, therefore, that cycloheximide has little influence upon the initial wave of infectious progeny production that

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2

TEMPERATURE SHIFT OF LA334 AFTER CYCLOHEXIMIDE BLOCK Sam- Time of cycle- Time at per- Virus ti- Percentage heximide missive ter (FFU/ of control Pie titer after treatme& temperaml) shift?’ bin) ture after shift? (rain) 1 2 3 4 5 6 7

None none -60 to +60 -5 to +60 -15 to +60 -30 to +60 -60 to +60

0 to +60 None None 0 to +60 0 to -t60 0 to +60 0 to +60

8 x lo4 6 x 10’
100 0.75 co.02 37 37 11 1.3

for a CEC were infected with LA334, maintained two passages at 35”, shifted to 42”, and maintained a further 2 days prior to transfer and plating in 60-mm Falcon plastic culture dishes at 42”. The cultures indicated received cycloheximide (5 pg/mll with a medium change 48 hr after transfer, and were shifted at various times after drug treatment from 42” back to 35”. The time periods inciated by a negative were before the shift was performed at 0 min. b The experiment was performed using a warm table (Colora Messtechnik; Larch, West Germany) maintained at 42” with a circulating water bath in a warm room with an ambient temperature of 40.5”. At the time of shift the cultures were removed to a 35” incubator. All samples were maintained with approximately 10% CO, atmosphere using sealed polyethylene bags (Vogt and Harris, 1974). Harvests for assay of virus released after shift were taken at +60 min and frozen at -80”. Samples 2 and 3 were harvested at +60 min, but remained during the entire course of the experiment on the warm table at 42” and were not shifted. (I The titer of released virus was determined in a focus assay and the results are expressed as focusforming units (FFUYml. ” The results are expressed as the percentage of the yield observed after shift of sample 1, which received no cycloheximide.

occurs with LA334 during the first hour after shift. Further experiments (data not shown) indicated that during the succeeding hours after shift, the inhibitory effect of cycloheximide becomes increasingly evident. Disturbance in the Course of Virus Maturation at the Nonpermissive Temperature The rapid onset of the production of infectious virus after shift of LA334-infected cells to 35” suggested that partially assembled viral structural intermediates might

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preexist in cells maintained at 42”. Electron microscopic investigation has revealed that in fact there is an accumulation of such structures at 42”. From Table 3 it is apparent that LA334-infected cells grown at 42” exhibit almost lo-fold more budding particles than are observed in sister cultures that had been shifted for 6 hr to 35” before processing for electron microscopy. This would suggest either that the interval during which the intracellular viral structural components remain partially assembled, and hence visible to electron microscopic observation, must be significantly longer at 42” than at 35”, or alternatively, that some lo-fold more viral particles are produced in a given time interval . TABLE

3

RELATIVE NUMBER OF BUDDING VIRUSES IN LA334 AND B77 Wt-INFECTED CELLS AT 35 AND 42"" Sample

LA334 B77 wt

Temperature of incubation (“1

Cell sections examined

42 35 42 35

236 260

93 119

Budding particles observed 37 4 3 2

(1 CEC were infected with LA334 or B77 wt and maintained for three passages at 35” prior to shift to 42”. Cells were maintained a further two passages at 42”, and at 90 hr after the final transfer, sister plates of each type were separated, half being shifted to 35”. After 6 hr of further incubation at either 42 or 35”, samples were harvested for infectivity assays, and the cells were rapidly fixed with 25% glutaraldehyde, as described in Materials and Methods. h Virus particles were observed in thin sections of embedded cells. The number of cell sections examined indicates the number of cell cross-sections screened for the presence of budding or released virus particles. In some cases cell sections obtained from the same cell at different planes were examined, but an effort was made to examine sections from different cells. c Budding virus particles were recognizable as electron dense aggregates of viral core material under the cell plasma membrane. With LA334-infected cells at 42”, these structures were often markedly atypical, but were readily distinguishable as virus specific. Where, as shown in Fig. 1B and C, budding particles appeared to be composed of sufficient core materials for more than one virus particle, the structure was in any case scored as a single budding viral structure.

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FRIIS

Previous experiments measuring the rate of release of radioactively labeled noninfectious physical particles from LA334infected cells (Friis et al., 1971) had indicated that little (less than 10% of the 35” yield) particle production occurred with cells incubated at the nonpermissive temperature. In the present experiments performed to examine this question, two improvements were made in the experimental design: (1) cells were infected and passaged at 35” until confluent transformation was microscopically visible, and then shifted to 42” prior to the determination; (2) radioactive labeling was accomplished in a pulse-chase experiment over an interval of 2 hr so that defective, structurally unstable particles would have a higher probability of being detected. Table 4 shows that in an experiment with these modifications, the yield of physical particles from LA334-infected cells at 42” was some 70% of the yield at 35”. On the basis of these results, it seems reasonable to conclude that LA334 produces a substantial yield of noninfectious virus particles at 42”, but that this yield is still less than the total yield observed in sister cultures incubated at 35”. Since there are many more budding virus particles observed at 42” than at 35” (Table 3), it seems probable that these accumulate at the nonpermissive temperature. After examination of electron micrographs taken of LA334-infected cells at 42”, a diversity in the morphology of the budding structures became apparent. Figure 1 shows three representative structures, each of which was scored as single viral budding structure. The crescentshaped core shown in panel A is indistinguishable from a normal budding avian Ctype virus particle. Panels B and C show budding structures which are, however, aberrant, as no such structures were observed with the B77 wt at 42 or 35”, or with the mutant at 35”. These atypical budding structures, which represent about 80% of the total observed, appear to be multiple buds, as in panel B, where it seems that the amount of core material is greater than the complement of a single virus, or malformed buds (panel C), where the core

ET AL. TABLE

4

RELEASE OF INFECTIOUS VIRUS AND VIRUS PARTICLES BY LA334 AT THE NONPERMISSIVE TEMPERAT] RE Sample Counts per Relative minute m yield of temreleased virus parviru@ ticlesh perature , (42”/35”) B71 wt 42” 35

5.1 x lo” 1.2 x lo;’

4.3

7 x 10” 4 x lo”

1.8

1.7 x lo” 2.4 x lo”

0.73

1 x 103 3 x lo”

0.0033

LA334 42 35”

‘I CEC were infected with B71 wt or LA334 and maintained through three passages at 35” before being shifted and maintained a further two passages at 42”. Sister plates were used for infectivity and radioisotopic-labeling experiments. For determination of released virus particles, cultures were pulse labeled with [3”S]methionine as described in the legend for Table 1, except that the total chase interval was 105 min in this experiment, and the samples referred to as “35”” were shifted immediately after the pulse to 35”. The virus-specific radioactivity was measured after virus was purified by sedimentation through a 20 to 50% sucrose gradient to equilibrium at a density of 1.15 to 1.16 g/cm3. Cultures were maintained at the indicated temperatures as described in the legend to Table 2. ’ The relative yield of radioactive virus particles or infectious virus was calculated by determining the ratio of the values at 42” to those at 35”, respectively. These values are underlined to emphasize the fact that they are derived. ’ Infectious virus in the supernatants was determined with a focus assay. Samples for the assay were taken 24 hr after the final medium change, and for the 35” samples, 2 hr after shift from 42 to 35”.

material seems uncondensed and diffuse, as if a normal core was elongated to extend along a broader front of cell membrane than is usual. Production of Viral Envelope Glycoprotein at the Nonpermissive Temperature Several findings had indicated that the expression of at least the viral envelope glycoprotein, gp85, is somehow deficient in LA334-infected cells at 42”: Owada and Toyoshima (1973) reported the failure of LA334 to act as a helper virus for the defective Bryan high titer strain of Rous sarcoma virus, and Katz and Vogt (1971) de-

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FIG. 1. Electron micrographs are presented showing budding virus particles observed in LA 334-infected cells at 42”: (a) Shows a normal-appearing virus bud; (b) and (c) illustrate atypical viral budding structures. The bar indicates 1000 A.

scribed an experiment which showed that after a shift to the permissive temperature, newly synthesized gp85 was preferentially incorporated into virus as compared to the other major viral structural proteins. Experiments were therefore performed to examine the expression of the viral envelope antigens on the surface of LA334infected cells at 35 and 42” using hybrid antibody immunoferritin electron microscopy. The hybrid antibody had dual specificity, to chicken IgG and to ferritin, and was reacted with cells which had been pretreated either with subgroup C-specific chicken antiserum, or with subgroup Aspecific chicken antiserum which served as

a control for the type specificity of the reaction. Figure 2 presents electron micrographs obtained in this study with the mutant grown at the nonpermissive temperature. Panels A and B show morphologically normal forms, a mature virus particle and a budding particle, which are specifically stained with the hybrid antibody; the amounts of ferritin bound to these structures are comparable to the amounts observed bound to similar particles produced by cells grown at the permissive temperature (micrographs not shown). Similar preparations stained with chicken antisubgroup A antiserum exhibited only a low background level of visible ferritin,

266

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FIG. 2. The localization of viral envelope antigens in immunoferritin electron micrographs is presented: (a) Shows extracellular released virus, (b) a normal-appearing virus bud, and (cl and (d) different types of atypical virus budding structures. An indirect hybrid antibody procedure was employed, using chicken antiB77 antiserum and rabbit hybrid antibody with dual specificities against chicken IgG and horse spleen ferritin. The bar indicates 2000 A.

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thus confirming the type specificity of the observed staining. Panels C and D illustrate the atypical budding structures which with the mutant-infected cells at the nonpermissive temperature account for 80% of the viral budding structures observed. Panel C exhibits specific staining while panel D shows only a background level of ferritin binding. Though, of course, a quantitative indication of the amounts of antigen present is impossible with this technique, in general it was found that the normal-appearing virus budding sites exhibited strong specific staining, whereas the aberrant viral budding structures entirely lacked, or showed reduced staining. A quantitation of cell-associated gp85 is possible since a rabbit antiserum prepared against highly purified Pr-C gp85 immunogen is available (Halpern et al., 1974). An indirect immunoprecipitation procedure employing this antiserum in excess

10

20

30

40

50

60

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allows quantitative recovery of gp85 from the cell lysate (M. S. Halpern, unpublished observations). The results obtained when this antiserum was used in indirect immunoprecipitation with lysates of “H-amino acid- and 3H-fucose-labeled cells are shown, respectively, in Figs. 3 and 4. All the immune precipitates, analyzed as shown in each figure, were prepared with volumes of lysates containing equal levels of TCAprecipitable radioactivity. Nonspecifically precipitating proteins, designated a,, a,‘, a2, and a3 are observed in the electropherograms of all the immune precipitates prepared with amino acid-labeled lysates. With the exception of a2, which probably represents actin (Fleissner and Tress, 19731, the origin of these proteins is unclear, but as they possess a different electrophoretic mobility than viral gp85, their appearance in the gels does not impede the analysis of gp85 synthesis. As detailed in Fig. 3, it is apparent that amino acid-la-

70 so FRACTION

10 20 (Zmml

30

40

50

60

70

i

FIG. 3. Electrophoreses on 5% SDS polyacrylamide gels of anti-g-p85 immune precipitates prepared with volumes of lysates of YH-amino acid-labeled cells containing 2 x IO6 trichloroacetic acid (TCAJ-precipitable cpm. (A) LA334-infected cells maintained at 42”. (B) LA334-infected cells maintained at 35”. (Cl B77 wtinfected cells maintained at 42”. (D) Uninfected cells maintained at 42”. ‘“C-Amino acid-labeled B77 wt was coelectrophoresed in all gels as marker. The pattern is shown in (A) and the position of viral gp85 and p27 are indicated in all panels. Electropherograms of immune precipitates prepared with infected cells and normal rabbit sera were indistinguishable from the pattern shown in (D).

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ET AL.

FRACTION

(2mm)

FIG. 4. Electrophoreses on 5% polyacrylamide gels of anti-gp85 immune precipitates prepared with cpm. The symbols and volumes of lysates of 3H-fucose-labeled cells containing 5 x lo4 TCA-precipitable panels are the same as for Fig. 3. Electropherograms of immune precipitates prepared with infected cells and normal rabbit sera were indistinguishable from the pattern shown in CD).

beled gp85 is present in lysates of LA334infected cells maintained at both 35 and 42” in amounts comparable to the level of gp85 in B77 Lot-infected cells. From the results of Fig. 4, it is also apparent that the gp85 in LA334-infected cells labels with fucose and, hence, is glycosylated at both temperatures. In addition, a small peak of fucose label that migrates with a similar electrophoretic mobility to gp37, the minor glycoprotein of avian tumor virus, is also observed (e.g., fractions 47-54, Fig. 4A). This peak is not detected with amino acid-labeling because it is obscured by the nonspecifically precipitating protein a2 which also comigrates with gp37. Although the evidence is not conclusive, we would tentatively assume that the peak detected with fucose labeling does in fact represent gp37 since recent evidence indicates that gp85 and gp37 share common antigenic determinants that are recognized by the antigp85 serum (Dr. D. Bolognesi, personal communication). In contrast to the findings obtained with hybrid antibody immunoferritin electron microscopy, which suggested a failure of interaction between the viral envelope glycoproteins and the aberrant viral budding structures, the biochemically characterized gp85, and probably also gp37, ob-

tained from immune precipitations of whole cell lysates, showed no detectable defect either in quantity or in mobility on SDS polyacrylamide gel. Further investigation of the expression of viral envelope glycoproteins was therefore undertaken using a biological approach. An interference test of greater sensitivity than in previous studies was performed taking advantage of the fact that avian leukosis viruses can rescue synthesis of infectious avian sarcoma virus from LA334-infected cells at 42”. If infected cells produce enough envelope glycoproteins at 42” to establish interference against superinfection by other subgroup C viruses, then no rescue by avian leukosis viruses of subgroup C (RAV-49) should occur although rescue by viruses of other subgroups such as A (RAV-3) should proceed. Table 5 indicates that a substantial rescue of LA334 can be obtained with the homologous subgroup, but that rescue by the heterologous subgroup is lo-fold more efficient. Two other temperature-sensitive mutants are shown for control purposes. La338, defective for an early, and perhaps also for a late replication function (Wyke, Molling, and Friis, unpublished observations), exhibits no interference and is equally well rescued by avian leukosis viruses of either

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TABLE 5 INTERFERENCE AGAINST RESCUE BY AVIAN LEUKOSIS VIRUSESP Mutant

virus

(subzrouu) I -

LA334 (C) LA338 (0 LA672 (A)

Rescuing avian leukosis virus (subgroup)

None RAV-3 (A) RAV-49 (C) None RAV-3 (A) RAV-49 (0 None RAV-3 (A) RAV-49 (C)

Virus titer at 42” (FFU/ml)b

4 6 2 5 9 9 4 4 7

x x x x x x x x x

102 lo4 103 103 104 104 103 103 l@

Virus titer at 35” (FFUlml)b

1 3 1 1 2 1 2 2 1

x x x x x x x x x

lo” 1P 105 10” 106 10” 10” 10” 10”

Rescue: Relative amount of virus yield’ (42”/ 35”) 0.004 0.2 0.02 0.005 0.05 0.09 0.002 0.002 0.7

Relative rescue at 42”” het. gkyp subgroup 22

0.5

&5cJ

n CEC were infected with the various mutants and transferred once at the permissive temperature. A shift to 42” was performed 24 hr later, followed by a further transfer into 60-mm Falcon plastic culture dishes so that individual samples could be obtained from sister cultures. Immediately after the final transfer, half of the plates were shifted back to 35”, and groups of plates at each temperature received either no superinfecting avian leukosis virus, RAV3 (m.o.i. = 51, or RAV-49 (m.o.i. = 3). Twenty-four hours after superinfection, cultures were medium-changed, and 48 hr after superinfection, samples were harvested for assay. b The samples were assayed in a focus test and the results are expressed as focus-forming units (FFU)/ml. c Equivalent samples (for example, LA334: RAV-3 superinfected) were compared for the release of infectious progeny at permissive and nonpermissive temperatures, and the data were expressed as a ratio. According to the results obtained with samples in the absence of superinfecting avian leukosis viruses, a value of 0.01 or greater for the relative amount of virus yield would appear to represent a significant rescue for any of the mutants tested. The maximum degree of rescue obtainable varies widely with the mutant. ” The rescue obtainable for a particular mutant with avian leukosis viruses of two different subgroups is expressed as a ratio of the degree of rescue seen with the heterologous (bet.) subgroup to that observed with the homologous (horn.) subgroup. This data represents the extent to which interference expressed by an individual mutant at the nonpermissive temperature can inhibit rescue of the mutant with an avian leukosis virus; therefore, values near 1 indicate absence of effective interference to avian leukosis virus superinfection, and high values, such as 350 observed with LA672, suggest a strong interference existing at the nonpermissive temperature. The values are underlined to stress their derivative character.

subgroup. LA672, a late mutant that produces large amounts of noninfectious particles lacking in reverse transcriptase activity (Friis and Hunter, 1973; Friis et al., 1975), established a strong interference to other subgroup A viruses, and was much more efficiently rescued by avian leukosis virus of subgroup C. Other rescuing leukosis viruses tested, subgroup A and C, behaved similarly to RAV-3 and RAV-49 with each of the mutants (data not shown). The degree of interference against superinfection by subgroup C viruses exhibited by LA334-infected cells at 42” was therefore an intermediate case, 5either as strong an interference as seen with LA672 nor a lack of interference as seen with LA338.

DISCUSSION

Because of the extremely rapid reversal of the block to replication observed with LA334 after temperature shift, we performed experiments to examine again the question of whether new protein synthesis was needed after shift for the burst of infectivity. In contrast to the experiment of Katz and Vogt (1971) in which cycloheximide was added 2 hr prior to the shift, we found that addition of the drug as little as 5 min before the shift was adequate to inhibit new viral protein synthesis, but not to inhibit release of infectious LA334. This result we have interpreted as suggestive that a pool of structural precursor exists which undergoes irreversible inactivation during the course of its normal proc-

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essing if maintained at the nonpermissive temperature. It is this pool which supplies functional protein when its processing is allowed to take place at the permissive temperature after shift. Electron microscopic examination has revealed the presence in LA334-infected cells of atypical viral budding structures. Since biochemical studies of these structures have not yet been carried out, it is impossible to know if they contain defective viral proteins; morphological characteristics are, however, sufficient to conclude that these structures are not part of the pathway of normal assembly with Ctype RNA tumor viruses. Their existence may derive from aberrant assembly of defective viral proteins resulting from incorrect processing of a temperature-sensitive assembly of precursor, or from partial structural protein precursors. It should be noted, however, that even the morphologically normal-appearing budding virus particles which were observed at a frequency of about 20% must also possess some structural defect since otherwise the titer of infectious virus observed at 42” would be much higher. In all probability, the hypothesis that a defect in the viral envelope glycoproteins of LA334 is the site of the essential lesion for replication, is false. Biochemical immunoprecipitation procedures were not able to detect any quantitative or qualitative deficiencies in intracellular viral envelope glycoproteins, though in these experiments there can be no certainty that gp37 was quantitatively assayed. Interference studies indicated that some functional viral envelope glycoprotein is present at the cell surface at the nonpermissive temperature, but suggested as well that the expression of these glycoproteins, or their function, is somewhat reduced. Furthermore, in contrast to the findings of Owada and Toyoshima (19731, successful helper activity for the Bryan high titer strain of Rous sarcoma virus, indicating the presence of functional viral envelope glycoprotein, was found with LA334-infected cells at the nonpermissive temperature (Friis, unpublished observations). Finally, data have been presented to show

ET AL.

that while some normal-appearing budding particles exhibit specific staining with hybrid antibody immunoferritin procedures employing type specific or neutralizing antibody, 80% of the viral budding structures demonstrated no staining or reduced specific staining. The accumulation of atypical viral budding structures may be readily explained by the hypothesis that LA334 bears a genetic lesion causing the synthesis of a defective viral core protein. Such a hypothesis has the advantage that a common precursor of the viral internal proteins (Vogt and Eisenmann, 1973) is known which exhibits a half-life of about 30 min (Vogt et al., 1975). Strong support for a hypothesis of a defective viral core protein in LA334 has been provided by studies of the protein composition of noninfectious virus particles produced at the nonpermissive temperature (Hunter et al., 1976; Rohrschneider et al ., In press, Virology), which have shown greatly reduced amounts of the major internal protein, ~27, and the appearance of a new structural protein, ~23. The fact that LA334 particle production continues at the nonpermissive temperature in spite of such a core defect is signiflcant, and suggests that the precision of assembly required for budding, and even release, is far from stringent. A temperature-sensitive mutant of Moloney murine leukemia virus, ts 3, which exhibits behavior similar to that of LA334, has been reported (Wong and McCarter, 1974). Significantly, this mutant, which is suspected of being defective in an envelope glycoprotein, fails to release physical viral particles at the nonpermissive temperature (Dr. P. Wong, personal communication). The influence of the probable viral core defect with LA334 on the localization of the viral envelope glycoproteins in the host cell membrane is most interesting. Reduced expression of viral envelope glycoproteins at the cell surface, as measured by the hybrid antibody immunoferritin electron microscopy and by the interfere:ice tests, presumably is a result of the atypical assembly of the viral cores. This finding implies that viral envelope formation at the cell membrane is dependent on

DEFECTIVE

MATURATION

an adjacently located viral core structure. Such an interdependence has been demonstrated for the assembly and budding of Semliki Forest virus (Garoff and Simons, 19741, although in that system, the process is believed to begin upon a single glycoprotein molecule, already located in the cell membrane, which serves as the nucleus for condensation of both core and additional glycoprotein molecules. The existence of the Bryan high titer strain of Rous sarcoma virus, a deletion mutant which fails to synthesize the viral glycoprotein gp85 (Kawai and Hanafusa, 19731, but which nevertheless produces large yields of noninfectious particles, makes any reciprocal dependence on viral envelope glycoproteins for core assembly in RNA tumor viruses uncertain. It is likely, however, that LA334 and other similar mutants may play a useful role in the study of the assembly and budding processes of RNA tumor viruses. ACKNOWLEDGMENTS The authors would like to thank Dr. Heinz Bauer for many useful discussions during the course of this work, and for critical reading of the manuscript. This study was supported by the Sonderforschungsbereich 47 of the Deutsche Forschungsgemeinschaft, by the Robert Koch Institut, and by Grants No. CA16047 and No. CA-10815 from the National Cancer Institute, U.S.A. REFERENCES E., and TRESS, E. (19731. Chromatographic and electrophoretic analysis of viral proteins from hamster and chicken cells transformed by Rous sarcoma virus. J. Viral. 11, 250-262. FRIIS, R. R., TOYOSHIMA, K., and VOCT, P. K. (19711. Conditional lethal mutants of avian sarcoma viruses. I. Physiology of ts 75 and ts 149. Virology 43, 375-389. FRIIS, R. R., and HUNTER, E. (19731. A temperature sensitive mutant of Rous sarcoma virus that is defective for replication. Virology 53, 479-483. FRIIS, R. R., MASON, W. S., CHEN, Y., and HALPERN, M. S. (19751. A replication defective mutant of Rous sarcoma virus which fails to make a functional reverse transcriptase. Virology 64,49-62. GELDERBLOM, H., BAUER, H., and GRAFT, T. (1972). eel!-surface antigens induced by avian RNA tumor viruses: Detection by immunoferritin technique. Virology 47, 416-425. GELDERBL~M, H., OGURA, H., and BAUER, H. (1974). FLEISSNER,

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On the occurrence of oncornavirus-like particles in HeLa cells. Cytobiologie 8, 339-344. GAROFF, H., and SIMONS, K. (19741. Location of the spike glycoproteins in the Semliki Forest virus membrane. Proc. Nat. Acad. Sci. USA 71, 39883992. HALPERN, M. S., BOLOGNESI, D. P., and LEWANDOWSKI, L. J. (19741. Isolation of the major viral glycoprotein and a putative precursor from cells transformed by avian sarcoma viruses. Proc. Nat. Acad. Sci. USA 71, 2342-2346. H~MMERLING, U., AOKI, T., DE HARVEN, E., BOYSE, E. S., and OLD, L. J. (1968). Use of hybrid antibody with anti-yG and anti-ferritin specificities in locating cell surface antigens by electron microscopy. J. Erp. Med. 128, 1461-1473. HANAFUSA, H., MIYAMOTO, T., and HANAFUSA, T. (1970). A cell-associated factor essential for formation of an infectious form of Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 67, 314-321. HUNTER, E., HAYMAN, M. J., RONGEY, R. W., and VOGT, P. K. (1976). An avian sarcoma virus mutant which is temperature sensitive for virion assembly. Virology 69, 35-49. KATZ, E., and VOGT, P. K. (1971). Conditional lethal mutants of avian sarcoma viruses. II. Analysis of the temperature sensitive lesion in ts 75. Virology 46, 745-753. KAWAI, S., and HANAFUSA, H. (1973). Isolation of a defective mutant of avian sarcoma virus. Proc. Nat. Acad. Sci. USA 70, 3493-3497. OWADA, M., and TOYOSHIMA, K. (1973). Analysis on the reproducing and cell-transforming capacities of a temperature sensitive mutant (ts 3341 of avian sarcoma virus B77. Virology 54, 170-178. RUBIN, H. (1960). A virus in chick embryos which induces resistance in vitro to infection with Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 46, 1105-1119. TOYOSHIMA, K., and VOGT, P. K. (1969). Temperature sensitive mutants of avian sarcoma virus. Virology 39, 930-931. VENABLE, J. H., and COGGESHALL, R. (1965). A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407-408. VOGT, P. K., TOYOSHIMA, K., and YOSHII, S. (19701. Factors promoting avian tumor virus infections. In “Defectivite’ Demasguage, et Stimulation des Virus Oncogenes,” pp. 229-238. Int. Symp. Tumor Viruses, 2nd, Royaumont. VOGT, P. K., WEISS, R. A., and HANAFUSA, H. (1974). Proposal for numbering mutants of avian leukosis and Sarcoma viruses. J. Viral. 13, 551554. VOGT, P. K., and HARRIS, P. (1974). Use of plastic bags to maintain a humidified atmosphere for animal cell cultures. Appl. Microbial. 27, 618-619. VOGT, V. M., and EISENMAN, R. (1973). Identifica-

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tion of a large polypeptide precursor of avian oncornavirus proteins. Proc. Nut. Acad. Sci. USA 70, 1734-1738. VOGT, V. M., EISENMAN, R., and DIGGELMANN, H. (1975). Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide. J. Mol. Biol., In press.

ET AL WONG, P. K. Y., and MCCARTER, J. A. (1974). Studies of two temperature sensitive mutants of Moloney Murine leukemia virus. Virology 58,396-408. WYKE, J. A., and LINIAL, M. (1973). Temperature sensitive avian sarcoma viruses: A physiological comparison of twenty mutants. Virology 53, 152161.