Characterization of moloney murine leukemia and sarcoma viruses separated by isokinetic gradient centrifugation

VIROLOGY

105, 436-444 (1980)

Characterization of Moloney Murine Leukemia and Sarcoma Viruses Separated by Isokinetic Gradient Centrifugation M . S . KISSIL, P . K. Y . WONG,' P . H . YUEN, AND M . M . SOONG Department of Microbiology and School of Basic Medical Sciences, University of Illinois, Urbana, Illinois 61801 Accepted May 6, 1980

Isokinetic sucrose gradients were used for the separation of different strains of murine sarcoma viruses (MuSV) from their associated helper leukemia virus (MuLV) . Two viralspecific peaks as determined by reverse transcriptase assay were obtained after a mixture of MuSV produced by MuSV349 cells and Moloney MuLV were centrifuged in an isokinetic gradient . The slower sedimenting peak fraction was shown to contain MuSV by the presence of RNA species which are smaller in size (-2 .2 x 108 daltons) than the genomic size of MuLV RNA, a protein gel electrophoresis profile similar to that of MuSV, the ability of the virus to form foci in TB and NRK cells, and its inability to produce syncytia in XC assay . The faster sedimenting peak was shown to contain MuLV by the presence of mainly MuLV genomic size RNA (-3.2 x 108 dalton), a protein gel electrophoresis profile similar to that of MuLV, and the ability of the virus to produce syncytia in the XC assay . INTRODUCTION

MATERIALS AND METHODS

The replication of the known strains of MuSV requires the presence of a nontransforming helper virus . Thus, any given sarcoma virus population contains helper virus usually in excess of the sarcoma virus . Therefore, it would be very advantageous for studies of MuSV if the sarcoma virus can be separated from its helper virus . Various methods including gel electrophoresis (Semancik, 1966 ; Zweig et al ., 1976), isoelectric focusing (Korant and Lonberg-Holm, 1974), and sucrose density gradient centrifugation (Bassinet al ., 1971) have been used to separate viruses in mixed populations . Since both MuSV and MuLV have a density of 1 .15-1 .16 g/cc in sucrose density gradients, they are not separable by this method . This report demonstrates by biological and biochemical characterization of the fractionated virus preparation obtained using isokinetic sucrose gradient centrifugation that MuSV can be separated from MuLV .

Cells and viruses . TB cells, a thymus bone marrow cell line from CFW/D mice, and 15F cells, a murine sarcoma-positive, leukemia-negative (S'L -) cell line, have been described previously (Wong and Gallick, 1978) . MuSV349, a cloned cell line obtained from Dr . J. K. Ball, was isolated from TB cells infected with Moloney MuSV (Ball et al ., 1973) . MuSV349 cells produce MuSV in great excess over MuLV (1000 :1) as determined by biological assay . The virus particles produced by MuSV349 are able to transform TB cells, but are unable to replicate in de novo infections in the absence of MuLV. Other cell lines included XC cells, derived from a rat tumor originally induced by the Prague strain of Rous sarcoma virus (Svoboda et at ., 1963), obtained from Dr . J . W . Hartley, and NRK, a normal rat kidney cell line, obtained from Dr . R. H . Bassin. All cell lines were maintained as previously described (Wong and McCarter, 1974) . Harvey murine sacroma (HaMuSV) and

' To whom requests for reprints should be addressed . 0042-6822/80/120436-09$02 .00/0 Copyright io 1980 by Academic Press, Inc. Al rights of reproduction in any form reserved .

436



SEPARATION OF MuSV AND MuLV

associated Moloney MuLV were obtained from Dr . E . H . Chang . Kirsten murine sarcoma (Kirsten murine leukemia)/NIH was obtained from NCI resource, and P521 M,MuSV (feline leukemia virus) was obtained from Dr. P . Fischinger . MuLV and ts3 were grown in TB cells and MuSV were obtained from MuSV349 cells . Virus assay . Culture fluids containing MuLV and MuSV were assayed as previously described by Wong and McCarter (1974) . Assay for MuSV was done by focus formation in TB cells . Briefly, 1 .0 x 10 5 TB cells were plated in each 60-mm plastic culture dish . Twelve to sixteen hours later the medium was removed and 0 .4 ml of virus diluted with serum-free medium containing 28 gg/ml DEAE-dextran was added and allowed to adsorb for 30 min . Complete medium was added and the cells were grown at 37° . Foci were counted 3-4 days postinfection. Assay of focus-forming virus on NRK cells was done as described by Parkman et al . (1970) . TB-XC assays were performed as described by Wong et al . (1973) . Isokinetic gradients . Isokinetic gradients, prepared as described by Noll (1967), were employed for the virus separation . A simple apparatus consisting of a magnetic stirrer, burette, and mixing chamber was used (Noll, 1967) . A volume of 20 ml of 25% (w/w) sucrose in THE buffer (10 mM TrisHCI, pH 7 .4 ; 100 mM NaCl; 1 mM EDTA) was placed in the mixing chamber . The burette was filled with 50% (w/w) sucrose in THE buffer and the gradient was poured into cellulose nitrate centrifuge tubes ("hs x 3'/z in .) . Samples, 0 .1-0 .5 ml, were layered on top of the gradients and centrifuged at 41,000 rpm for 90 min in a Beckman SW 41 rotor . Gradients were fractionated with an ISCO Model 640 fractionator and scanned for absorbance at 280 nm with an ISCO UA5 recorder. RNA extraction and electrophoresis . Virus was pelleted and resuspended in 1 ml of THE buffer containing 50 pg of yeast tRNA as carrier . Sodium dodecyl sulfate (SDS) was added to a final concentration of 2% (w/v) and the mixture incubated at room

437

temperature for 20 min . The resultant mixture was extracted two times with TNE-saturated phenol :cresol:8-hydroxyquinoline :10 :1 :0 .01 . The extracted RNA was precipitated from the final aqueous phase by the addition of 2 vol of -20° ethanol, and allowed to stand overnight at -20° . The resultant RNA was pelleted and lyophilized . Electrophoresis was carried out in 3.5% polyacrylamide/formamide gels as described by Duesberg and V ogt (1973) . After electrophoresis, the gels were sliced with a Mickle gel slicer, solubilized, and counted in a liquid scintillation counter . SDS-polyaerylamide gel electrophoresis . Gel electrophoresis was done in a linear 7 .5-17 .5% gradient slab gel using the discontinuous buffer system of Laemmli (1970) . Samples dissociated in 62 .5 m1V Tris (pH 6 .8) containing 2 .3% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, and 10% (wlv) glycerol were boiled for 1 min and electrophoresed at 10 mA/gel for 16 hr . The gels were then stained with 0 .25% Coomassie brilliant blue (CB) in a 10% acetic acid20% isopropanol mixture and destained progressively in 0 .037% CB in 10% acetic acid-10% isopropanol, 0 .03% CB in 10% acetic acid, and finally in 10% acetic acid . ['H] Uridine and 22P labeling of virus . Virus-producing cells were grown in 150em' flasks . At approximately 2 days before confluency, the medium was replaced with phosphate-free MEM containing 10% dialyzed fetal calf serum and incubated for 12 hr . At this time the medium was replaced with fresh medium containing [3H]uridine (67 gCi/ml) or 32P, as H,'2PO, (80 pCilml), and incubated for an additional 12 hr . Medium was then harvested at 2- to 3-hr intervals for 24-48 hr and virus purified . Reverse transcriptase assay . Reverse transcriptase activity was determined as described by Wong et al . (1977) . Aliquots (30 pl) were assayed in 100-µl reaction mixtures containing 50 mM Tris-HCI (pH 8.0), 60 mM KCI, 1 mM MnCl,, 2 .5 mM DTT, 0 .05% Triton X-100, 0.04 OD,s„ units of polyribocytidylic acid :oligodeoxyguanylic acid 12-18 (rC :dG), and 0 .2 mM [3H]dGTP (10 pCi/mmol).



43 8

KISSIL ET AL .

FIG . 1 . Transmission electron micrograph of a representative thin section of ts(MuLV)-infected 15F(A) and MuSV349(B) cells showing different size extracellular immature C-type virus particles . Representative particles of particles ranging in size from 105-110 nm(a), 108-122 nm(b), 95-104 nm(c), and 82-85 nm(d) .

Transmission electron microscopy (TEM) . Virus-infected cells were prepared for electron microscopy as described by Yuen and Wong (1977a) . RESULTS

Electron Microscopic Studies on the Size of MuLV and MuSV In a previous study (Yuen and Wong, 1977a), it was demonstrated that the accumulation of virus particles on the cell membrane of ts3 (a temperature-sensitive mutant of Mo-MuLV)-infected TB cells at the nonpermissive temperature and their rapid release as immature C-type particles on temperature shift down provided a means for measuring the size of a large number of extracellular immature particles with minimal distortion . It can be shown using thin section electron microscopy that immature type C particles of varying sizes similarly accumulated on the cell membrane of 15F (S*L -) cells infected

with ts3 . On temperature shift down, these particles were also released as immature particles (Fig. 1A) . This observation suggested a means for determining the size of MuSV by comparing the size of particles produced by ts3-infected TB cells with those produced by ts3-infected 15F cells and virus produced by MuSV349 cells . Figure 2A is a histogram comparing the frequency of extracellular immature type C particles of varying size classes obtained from ts3infected 15F cells (Fig . 1A) and MuSV349 cells (Fig. 1B) . While the size of the particles produced by both cell lines extends more or less over a similar range, most of the virus produced by MuSV349 measured about 100 nm in diameter (Figs . 113c and 2Ac) . In contrast, the particles produced by ts3-infected 15F cells fell predominantly into two size categories, a smaller particle measuring about 100 nm in diameter (Figs . lAc and 2Ac) and a larger particle measuring about 108 nm in diameter (Figs . 1Aa and 2Aa) . Particles of 118-120 nm in diameter



439

SEPARATION OF MuSV AND MuLV

u

20

A

10

MuSV and MuLV indicated by electron microscopy suggested the possibility that these particles could be separated by using isokinetic gradients, a method designed to separate particles of similar densities based on their differing rates of sedimentation through a sucrose gradient .

b

V

Separation of MuSV and MuLV Particles Using Isokinetic Gradients

0

20

80

90

100 110 120

30

six. innonwlni FIG. 2 . Histogram showing the variation of the size of extracellular immature MuSV349, TB-MuLV, and progeny from ts3(MuLV)-infected 15F cells (which contain both MuLV and MuSV) . (A) A comparison of the size of MuSV349 (/) and virus progeny from ts3-infected 15F cells (30 min after shift from 39 to 34') (O) . (B) A comparison of the size of MuLV (U) and ts3 from TB cells 30 min after shift from 39 to 34° (O) . The different sized particles of Fig . 1 correspond to a-d in this figure .

(Figs . lAb and 2Ab) were also observed, but these were found to be multiploids, that is spherical particles apparently containing two sets of diploid genomes (Yuen and Wong, 1977a) . Figure 2B further shows that the particles produced by ts3-infected TB cells appear to consist of two distinct size classes : in one size class, most of the particles measured 104-110 nm in diameter while in the other most of the particles measured 116122 nm in diameter . Again the larger size particles were found to be multiploids . It was previously shown (Yuen and Wong, 1977a, b) that the high proportion of multiploid particles produced by ts3-infected cells was related to the accumulation of budding particles on the cell membrane at the nonpermissive temperature. In contrast, most of the particles of MuLV produced in TB cells measured 104-108 nm in diameter (Fig . 2B (shaded area)) . The above observations suggest that the size of MuSV particles may be somewhat smaller than that of MuLV. The size difference between

Purified Mo-MuLV grown in TB cells and MuSV produced by MuSV349 were centrifuged in separate isokinetic gradients, and a mixture of the two viruses (approximately equal quantity as determined by 0D zae nm) was centrifuged in a parallel gradient . The results of representative gradients of such an experiment are shown in Fig . 3 . Both MuSV and MuLV exhibited a single peak when centrifuged alone (Figs . 3A and B) . MuSV, which appeared to be smaller as determined by electron microscopy, exhibited a slower sedimentation rate with respect to MuLV . A mixture of the two viruses (Fig. 3C) centrifuged in a single gradient yielded two major peaks (which throughout the remainder of the paper will be referred to as peaks I and II) which corresponded in the location in the gradient to the individual peaks obtained when

wwnacraara iiii FIG . 3 . Isokinetic sucrose gradient purification of (A) MuSV349, (B) Mo-MuLV, and (C) a mixture of MuSV349 and Mo-MuLV .



440

KISSIL ET AL .

MuLV or MuSV particles were centrifuged alone (Figs . 3A and B) ; that the two peaks contained virus was demonstrated by reverse transcriptase assay (data not shown) . The specific identities of the virus in peaks I and II were further characterized biochemically and biologically . Characterization of Virus Contained in Peaks I and II In order to determine whether peaks I and II in the isokinetic gradients contain MuLV and MuSV, respectively, the RNA and protein profiles, and infectivities from each peak were examined . Viral RNAs . 32 P-Labeled Mo-MuLV and [3 H]uridine-labeled MuSV349 virus were pelleted and mixed . RNA extracted from an aliquot of the virus mixture before isokinetic gradient separation and from each peak (I and II) following isokinetic gradient separation (cf. Fig. 3) was analyzed by formamide-polyacrylamide gels as described under Materials and Methods . The results are shown in Fig . 4. Figure 4A shows the gel profile of the RNA extracted from a mixture of MuSV and MuLV before isokinetic gradient centrifugation . The [ 3H]uridine-labeled MuSV shows a major -2 .2 x 106 dalton and a minor -3 .2 x 10 6 dalton RNA species . The 32 P-MuLV RNA profile consists of a larger peak of -3 .2 x 10 6 daltons and smaller peak of --1 .9 x 106 daltons . The relative amounts of the 3 .2 x 10 6 and 1 .9 x 10 6 dalton RNAs varied between preparations, but the larger RNA invariably is the major species . It is possible that the 1 .9 x 10 6 dalton RNA species is the same subgenomic RNA species of MoMuLV described by Ball et al . (1979) . As shown in Fig. 4B, the virus obtained from peak I, following isokinetic gradient separation, exhibited an RNA profile identical to that of MuSV with <15% contamination from 32P-MuLV . In contrast the profile of the RNA extracted from virus in peak II (Fig. 4C) was identical to that of MuLV . The small amount of the larger MuSV RNA recovered from peak II accounted for less than 12% of the total MuSV RNA . Additional experiments examining the

3-

i

I

S 2

0 0

3D S1w Natsr

FIG . 4 . Formamide-polyacrylamide gel electrophoresis of RNA isolated from 1 3 H]uridine-labeled MuSV349 (0) and "P-labeled Mo-MuLV (0), (A) coelectrophoresis of pelleted MuSV349 and Mo-MuLV RNAs, (B) peak 1 RNA, and (C) peak II RNA .



44 1

SEPARATION OF MuSV AND MuLV TABLE 1 RELATIVE AMOUNTS OF p12E AND p15 IN MUSV, AND IN VIRUS RECOVERED FROM PEAK I AND

MuLV,

PEAK II AFTER ISOKINETIC SUCROSE GRADIENT SEPARATION OF A MIXTURE OF MUSV AND MuLV

Virus

Ratio pI2E/p15"

MuSV MuLV I 11

1 .34 0.79 1 .22 0.74

To determine the pl2E/p15 ratio the stained gels were scanned and areas under the p12E and p15 peaks were cut and weighed .

native virion RNA of peaks I and II also indicated a smaller RNA (40-50 S) associated with the slower sedimenting peak I as well as a larger RNA (55-70 S) associated with the faster sedimenting peak II (data not shown) . Viral proteins . The protein profiles of Mo-MuLV and MuSV349 visualized by SDS-PAGE are shown in Fig . 5. The protein patterns differ in the relative amount of the low molecular weight proteins, and the presence of additional bands in MuSV above p30 and in the Pr65 region (indicated by arrows) . The MuLV protein profile exhibited a greater amount of p15 relative to p12E (Table 1), whereas, the MuSV protein profile showed relatively more p12E than p15 (Table 1) . As shown in Fig. 5, the protein profile on the virus obtained from peak I is similar to that of MuSV, while the protein profile of the virus obtained from peak II is similar to that of MuLV . Biological assays . The identities of the virus contained in peaks I and II were further determined using infectivity assays . The TB and NRK focus assays were used to detect the presence of MuSV while the XC assay was used to detect the presence of MuLV. The results shown in Table 2 indicated that the number of XC-positive colonies produced by both pelleted MuSV and isokinetic gradient-purified MuSV was not above background levels, whereas TB

cells infected with pelleted MuLV or isokinetic gradient-purified MuLV when tested with the XC assay gave titers of 1 .8 x 10' and 4 .0 x 10' IU/ml, respectively . Virus from the slower sedimenting peak (peak I) did not produce XC-positive colonies whereas peak II was shown to contain 4 .2 x 10' IU/ml . This observation indicated that peak I was not contaminated by MuLV . Using the TB focus assay, titers of 4 .5 x 10" and 1 .0 x 10' FFU/ml were obtained from pelleted MuSV and isokinetic gradient-purified MuSV, respectively . No detectable foci were obtained either with pelleted or isokinetic gradient-purified MuLV . Virus from both peaks I and II produced foci in TB cells . However, there is a 20- to 25-fold difference in the MuSV titer between peaks I and II . A similar difference in titer was also obtained when virus from peaks I and II were assayed on NRK cells . Separation of other murine sarcoma and leukemia viruses . We have also used isoMLV

MSV

a

I

gp 70

pr 65

p 30

p15E p 12E p15 p12 P10

FIG . 5 . SDS-polyacrylamide gel electrophoresis of Mo-MuLV, MuSV349, and virus peaks I and peaks 11 separated from a mixture of Mo-MuLV and MuSV349 .



4 42

KISSIL ET AL. TABLE 2 DETERMINATION OF VIRUS INFECTIVITY BEFORE AND AFTER ISORINETIC GRADIENT SEPARATION

Indicator cells Virus

TB (FFU/mp

NRK (FFU/ml)

XC (IU/ml)

Pelleted MuSV349 Pelleted MuLV Isoldnetie gradient purified MuSV349 MuLV Peak 1 Peak II

4 .5 x 10' ND

1 .6 x 101 ND

ND' 1 .8 x 10'

1 .0 x 10' ND 3.2 x 10' 1 .4 x 102

3 .0 x 102 ND 1 .1 x 10' 5 .0 x 10'

ND 4 .0 x 10' ND 4 .2 x 10'

° Not detectable.

kinetic gradients to separate the following sarcoma-leukemia virus mixtures : Harvey sarcoma virus : Moloney leukemia virus (HaMuSV :Mo-MuLV), Kirsten sarcoma virus : Kirsten leukemia virus (KiMuSV : KiMuLV), and Moloney sarcoma virus: feline leukemia virus (M 1MuSV:FeLV) . As shown in Fig . 6, in all cases except for KiMuSV :KiMuLV mixtures (Fig . 6C), two distinct viral peaks were obtained . The faster sedimenting peak in the gradient fractionation of the mixture of HaMuSV and Mo-MuLV (Fig. 6B) and the faster sedimenting peak in the gradient fractionation of the mixture of KiMuSV and KiMuLV (Fig. 6C) corresponded in position to peak II of the gradient fractionation of MuSV349 from Mo-MuLV (Fig . 6A) . Figure 6D shows that the faster sedimenting peak in the gradient fractionation of FeLV from M 1MuSV moved at a slower rate than peak II in the gradient separating MuSV349 and Mo-MuLV (Fig . 6A) . The isokinetic gradient conditions for the separation of these various mixtures of MuSV and their associated helper virus may not necessarily be optimal . For comparison purposes we have employed the conditions optimized only for the separation of MuSV349 and Mo-MuLV (see Materials and Methods) . Figure 6 further shows that there is a greater variability in the sedimentation rates of the virus particles in the slower moving peaks suggesting that there is variability in the size of different MuSV .

Separation of MuSV from MuLV after rescue from 15F cells . To further illustrate the applicability of this technique, 15F cells were infected with MuLV and the resultant progeny virus harvested . The pelleted virus was layered on a 25-50% (w/w) isokinetic sucrose gradient and centrifuged as described . Figure 7B is representative of the observed separation . A smaller, slower sedimenting peak and a larger, faster sedimenting peak were obtained corresponding to peaks I and II separated from a mixture of MuSV349 and MuLV (Fig . 7A) . Preliminary SDS-PAGE analysis (data not shown) of virus in each peak (Fig . 7B) indicates distinct protein profiles corresponding to the differences seen with MuSV349 and Mo-MuLV (cf. Fig . 5) . DISCUSSION

Studies on mammalian sarcoma viruses have been greatly hampered by the inability to isolate sarcoma viruses completely free of helper virus . From our electron microscopic studies on thin section of extracellular immature type C particles, we observed that MuSV from MuSV349 cells and MuSV rescued from 15F cells are distinctly smaller than Mo-MuLV which suggest that MuSV particles may be smaller than MuLV . We, therefore, attempted to separate MuLV from MuSV using isokinetic sucrose gradients which were de-



443

SEPARATION OF MuSV AND MuLV

A

0.6

1

0.0 0 .9

><

J -

e

C

a0

U

a0 a0

en

53.0

Volume of CraAant Imll FIG. 6 . Isoldnetie sucrose gradient separations of (A) a mixture of MuSV349 and MuLV, (B) Harvey

sarcoma virus from Moloney leukemia virus, (C) Kirsten sarcoma virus from Kirsten leukemia virus, and (D) Moloney M,MuSV from feline leukemia virus .

signed to separate particles or molecules of similar densities based on their differing rates of sedimentation . In contrast to linear sucrose gradients, the design of the isokinetic gradient minimizes the effects of viscosity on the sedimentation of particles, thus allowing a constant sedimentation rate throughout the length of the gradient . Using this technique, we have demonstrated the utility of this technique for the separation of sarcoma viruses from their associated helper viruses . When MuSV produced by MuSV349, a cell line that produces sarcoma virus with no detectable MuLV by XC assay, was mixed with Mo-MuLV, two distinct peaks were obtained after isokinetic gradient centrifugation . Virus which sedimented slower in the gradient was demonstrated to be MuSV by the isolation from the virus fraction of RNA species which is smaller in size (2 .2 x 10 6 daltons) than MuLV genomic

RNA, the similarity of the protein profile of this virus to that of MuSV349, its ability to form foci in NRK and TB cells, and its inability to form XC-positive TB cell colonies . The faster sedimenting virus was identified as MuLV by the isolation from this virus fraction of mainly MuLV genomic size RNA (3 .2 x 106), by the similarity of the protein profile of this virus to that of Mo-MuLV, and by its ability to produce XC-positive TB cell colonies . The smaller, 1 .9 x 10 6 dalton, RNA observed in MuLV grown in TB cells is likely to be the same subgenomic RNA species, packaged within the virus, observed by Ball et al . (1979) . These results, in addition to the reduced amount of the --3 .2 x 10 6 dalton RNA species observed in peak I (Figs . 4A and B), indicated that the MuSV recovered after isokinetic gradient fractionation was reasonably free of helper virus and should prove to be useful for biochemical analysis . In addition, the virus purified using isokinetic gradients retained more infectivity than virus isolated using linear sucrose density gradients . While a 5- to 50-fold decrease in infectivity was obtained following isokinetic gradient centrifugation (cf. Table 2), a 100- to 1000-fold decrease in infectivity was obtained from virus after sucrose density gradient purification (data not shown) . Using the same conditions for the prepa-

Fir . 7 . Isokinetic sucrose gradient separations of (A) a mixture of MuSV349 and Mo-MuLV, and (B)

harvested virus from an infection of Mo-MuLV .

15F

cells with

KISSIL ET AL .

ration and centrifugation of the isokinetic gradients, we have also partially separated other murine sarcoma-leukemia mixtures . Our results indicate that while different strains of murine leukemia virus appear to sediment at about the same rate through the gradient, a much greater variability was observed for the sedimentation of the different strains of MuSV tested . This difference in sedimentation rate can be attributed to variability in the size of the different strains of MuSV. The faster sedimentation rate obtained by us for Kirsten MuSV compared to that of Harvey MuSV is consistent with the larger size of the RNA of Kirsten MuSV compared to that of Harvey MuSV reported by Maisel et al . (1977) . Although it is well established that the size of RNA of MuSV is smaller than that of MuLV, this is the first report demonstrating that the size of the MuSV particle is correlated to the size of the RNA . ACKNOWLEDGMENTS

KoRANT, B . D ., and LONBERG-HORN, K. (1974). Zonal electrophoresis and isoelectric focusing of proteins and virus particles in density gradients of small volume . Anal . Biochem . 59, 75-82. LAEMMLI, U . K . (1970) . Cleavage of structural proteins during assembly of the head of bacteriophage T4 . Nature (London) 272, 680-685 . MAISEL, J ., DINA, D., and DUESBERG, P. (1977). Murine sarcoma viruses: The helper-independence reported for a Moloney variant is unconfirmed ; distinct strains differ in the size of their RNAs . Virology 76, 295-312 . NOLL, H . (1967) . Characterization of macromolecules by constant velocity sedimentation . Nature (London) 215, 360-363. PARKMAN, R ., LEVY, J . A ., and TING, R . C . (1970) . Murine sarcoma virus : The question of defectiveness . Science 168, 387-389 . SEMANCIK, J . C . (1966) . Studies on electrophoretic heterogeneity in isomeric plant viruses . Virology 30, 698-704 . SVOBODA, J ., CHYLE, P., SIMKDvIC, D ., and HILGERT, I . (1963). Demonstration of the absence of infectious Rous virus in rat tumor XC, whose structurally intact cells produce Rous sarcoma when transferred to chicks. Folia Biol . (Prague) 9, 77-81 .

The authors wish to thank Dr . M . E . Reichmann and Dr . C . M . Bergholz for reviewing this paper. This investigation was supported by Public Health Service Research Grant CA 19723 and CA 17695 within the Virus Cancer Program of the National Cancer

WONG, P . K. Y., and GALLICK, G . E . (1978). Preliminary characterization of a temperature-sensitive mutant of Moloney murine leukemia virus that produces particles at the restricted temperature . J . Virol. 25, 187-192.

Institute .

WONG, P . K. Y ., MACLEOD, R . . CHANG, E . H . MYERS, M . W., and FRIEDMAN, R. M . (1977) . Theeffeet of interferon on de hove infection of Moloney murine leukemia virus . Cell 10, 245-252 .

REFERENCES BALL, J . K., DEKABAN, G. A., LOOSMORE, S . M ., CHAN, S . K ., and MGCARTER, J . A . (1979) . Subgenomic RNA in Moloney leukemia virus grown in lymphoid-derived cell lines consists primarily of a homologous viral RNA . Nucleic Acids Res. 7,1091 1108 . BALL, J . K ., MCCARTER, J . A., and SUNDERLAND, S . M . (1973) . Evidence for helper-independent murine sarcoma virus . I. Segregation of replicationdefective and transformation-defective viruses . Virology 56, 268-284 . BASSIN, R . H ., PHILLIPS, L . A., KRAMER, M . J ., HAAPALA, D . K ., PEEBLES, P . T., NOMURA, S., and FISCHINGER, P . J . (1971) . Transformation of mouse 3T3 cells by marine sarcoma virus : Release of viruslike particles in the absence of replicating murine leukemia helper virus . Proc . Nat . Aced. Sci . USA 68, 1520-1524 . DUESBEEG, P. H ., and VOGT, P. K . (1973) . Gel electrophoresis of avian leukosis and sarcoma viral RNA in formamide: Comparison with other viral and cellular RNA species . J. Virol . 12, 594-599 .

WONG, P . K . Y ., and MCCARTER, J . A. (1974) . Studies of two temperature-sensitive mutants of Moloney murine leukemia virus . Virology 58, 396-408 . WONG, P . K . Y ., Russ, L . J ., and McCARTER, J . A . (1973) . Rapid, selective procedure for isolation of spontaneous temperature-sensitive mutants of Moloney murine leukemia virus . Virology 51, 424-431 . YUEN, P . H ., and WONG, P. K . Y. (1977a) . A morphologic study on the ultrastructure and assembly of murine leukemia virus using a temperaturesensitive mutant restricted in assembly . Virology 80, 260-274 . YUEN, P. H ., and WONG, P . K . Y. (1977b) . Electron microscopic characterization of the defectiveness of a temperature-sensitive mutant of Mo-MuLV restricted in assembly . J. Virol . 24, 222-230 . ZWEIG, M ., BARRAN, S ., and SALZMAN, N . P . (1976) . Analysis of simian virus 40 wild-type and mutant virions by agarose gel electrophoresis . J. Virol . 17, 916-923.