Murine sarcoma virus defectiveness: Serological detection of only helper virus reverse transcriptase in sarcoma virus rescued from nonmurine S+L− cells

70, 313-323 (1976)

VIROLOGY

Murine Sarcoma Virus Defectiveness: Serological Detection of Only Helper Virus Reverse Transcriptase in Sarcoma Virus Rescued from Nonmurine S+LCells PAUL Viral

T. PEEBLES, Leukemia

BRENDA

and Lymphoma

Branch,

I. GERWIN, National

AND

EDWARD

Cancer Institute,

Accepted November

Bethesda,

M. SCOLNICK Maryland

20014

4, 1975

Murine sarcoma virus (MSV) defectiveness was studied by examining MSV rescued by RD- 114 virus superinfection of both dog and human heterologous host cells nonproductively transformed by the S+L- strain of Moloney MSV (S+L- cells). Biological assays showed that the rescued MSV pseudotype was present in excess over the RD-114 helper virus. The biologically determined virus ratio corresponded to that determined by virus nucleic acid hybridizations. Antisera distinguishing the DNA polymerase of murine type C virus from that of RD-114 virus demonstrated that the only detectable RNAdependent DNA polymerase present in the rescued MSV virions was that of RD-114 helper virus. No murine virus polymerase was detectable within the virions. Previous data have demonstrated that no murine-viral polymerase is associated with S+L- heterologous human, dog, and mink host cells. The work presented here demonstrates that cells producing infectious MSV and helper virions still do not express murine polymerase within released MSV virions. These findings suggest that heterologous host-cell control mechanisms are not preventing MSV polymerase expression in these cells. Instead, the rescued MSV virions acquire the polymerase protein from the rescuing helper virus. These data provide evidence that the MSV genome lacks the information necessary for polymerase, and requires for the production of infectious progeny the functioning of a helper virus replication gene set supplying at least viral envelope antigen(s) and virion core protein polymerase. INTRODUCTION

Moloney murine sarcoma virus (MMSV) can enter and transform murine and nonmurine cells in the absence of a helper virus (Aaronson et al., 1970; Bassin et al., 1970; Peebles et al., 1973a, 1975). M-MSV is, however, defective and unable to thereafter replicate infectious progeny from these cells. The nature of this defect(s) has been under investigation. It has been demonstrated that sarcoma virus-positive leukemia virus-negative (S + L-1 murine cells express detectable but quantitatively deficient murine-viral reverse transcriptase in noninfectious type C virions released into their culture supernatant fluids. The same MSV, when recloned in heterologous human, dog, and mink cells, fails to express

any murine-viral reverse transcriptase (Peebles et al., 1975b). Three possible explanations for this consistent difference have been proposed (Peebles et al., 1975b): (i) the MSV genome was altered or a mutant isolated when it was originally cloned in human cells; (ii) MSV codes for reverse transcriptase, but its expression is prevented in heterologous host cells; (iii) MSV lacks the gene for its own reverse transcriptase and this function is supplied by replicating, but nontransforming, helper virus. The first possibility, namely that a mutant deficient in polymerase had been isolated, has been eliminated by showing that when the MSV genome from S+ Lhuman cells is back passed into mouse cells, murine-viral reverse transcriptase is 313

Copyright All rights

6 1976 by Academic Press, Inc. of reproduction in any form reserved.

314

PEEBLES,

GERWIN

AND

SCOLNICK

again expressed in noninfectious virions medium cloning procedures (Godsby and released into culture supernatants of these Zipser, 1969). Normal F-445-l dog kidney S+L- mouse cells (Peebles et al., 1975b). cells and S+L- dog cells (S+L- DoCl,) The studies herein were initiated to de- have been described previously (Peebles et termine whether murine-viral polymerase al., 1975b). High containment culture could be detected in infectious MSV vi- methods with these cell lines have previrions rescued from heterologous host ously been described in detail (Peebles et S+L- cells. Since heterologous host cell al., 1971; Peebles et al., 1973a). control may have prevented expression of Viruses and virus assays. RD-114 virus murine reverse transcriptase in the ab- was harvested from filtered (0.45 pm) susence of replicating helper virus (Peebles pernatant culture fluids of RD-114 cells et al., 1975b), a burst of murine polymerase (McAllister et al., 1971). RD-114 virus was should be detected in MSV virions rescued biologically assayed by three separate by superinfection of these S + L - cells with methods. First, 0.5 ml of half-log dilutions a nonmurine helper virus. Instead, no mu- of the indicated virus stocks containing rine-viral polymerase was detected in RD-114 virus were applied to F-160-5 MSV rescued from both dog and human S+LHuA and S+Lmink clone, S+L- cells. The only serologically detect- (MiCl,) cells (Peebles, 1975a) in a manner able reverse transcriptase was that of the essentially as that described for MuLV in heterologous rescuing helper RD-114 vi- the mouse system (Bassin et al., 1971b). rus. Both virus stocks contained MSV in Disposo trays, FC-16-24TC (Bellco Glass, Inc., Vineland, N. J.) must be used in the excess of the associated helper virus. These findings, along with earlier data assay with F-160-5 cells for maintenance of demonstrating that a mutant MSV was a background monolayer. Foci induced by not being studied (Peebles et al., 1975131, the helper virus were counted at approxisuggest that the sarcoma virus is defective mately Day 10 when the monolayers were in information for its own viral polymer- confluent and focus-inducing units (FIU) ase. This core protein necessary for infec- per milliliter were calculated. tious transforming MSV progeny is apparThe second biological assay for RD-114 ently supplied by the rescuing helper vi- virus involved two steps. Five-tenths-milI-US. liliter aliquots of the same virus dilutions were applied to eight separate wells per dilution of Disposo trays containing MATERIALS AND METHODS DEAE-dextran (25 pg/ml) pretreated Cell lines and culture techniques. F-49-l S+L- HuA cells. At 6 days postinfection, human cells were derived as described supernatant fluids harvested from each (Peebles et al., 1973a) from a more contact- well were individually applied to Disposo inhibited single-cell clone of the AV-3 hu- tray wells containing lo4 DEAE-dextran man cell line (no original publication) ob- pretreated normal F-49-l human cells. Wells positive for foci from the RD-114 tained from the American Type Culture Collection (ATCC), Rockville, Md. AV-3 helper virus rescued MSV were counted 10 cells have some of the chromosome days later, and the helper virus replicating markers found in HeLa cells (Nelson-Rees units, VRU/ml, were calculated. The third biological assay method for et al., 1974). A focus-derived single-cell RD-114 virus likewise employed 0.5-ml aliclone of S+Lhuman cells (S+LHuACl,) transformed by MSV in the ab- quots of the same virus dilutions, applied this time to eight wells per dilution of sence of detectable leukemia-helper-type viruses has been previously described (Pa- Disposo trays containing DEAE-dextran pretreated normal F-49-l human cells. pageorge et al., 1974). A flatter variant line, designated as F-160-5 cells, was gen- After 8 weeks of subculturing, the cells were assayed for RD-114 p30 presence by erated from a single-cell clone of the original focus-derived S+L- human cells (Pee- complement fixation and radioimmunoassay, and the VRU/ml was calculated. bles et al., 1973a) using described liquid-

MSV

DEFECTIVENESS

Focus-forming units, FFU/ml, of the RD-114 virus pseudotype of MSV were assayed on F-49-l human cells. The addition of optimal helper virus is not required for “one-hit” titration kinetics of focus formation on these cells. However, RD-114 virus may be used at optimal concentration to enlarge the foci and facilitate counting (Peebles et al., 1973b). Noninfectious Ctype virus particles from S+ L - mouse cells were concentrated by ultracentrifugation of supernatant culture fluids as described (Bassin et al., 1971a). Double sucrose density-gradient purified Rauscher murine leukemia virus (R-MuLV), 10” virus particles/ml, was purchased from Electro-Nucleonics, Rockville, Md. Detection of MuLV group specific (~30)

and RD-114 determinants.

virus

The complement fixation (CF) assay for detection of virus gs-1 (p30) antigens using lowdose immunized guinea pig antisera has been described (Gilden et al., 1971). Radioimmunoassay for RD-114 virus p30 antigens has also been detailed (Parks and Scolnick, 1972). Chemicals. Poly(A), poly(dA), poly(C), and poly(dT) were obtained from Miles Laboratories, Inc., Kankakee, Ill.; and (dT),,-,, and (dG),,-,, were purchased from Collaborative Research, Inc., Waltham, Mass. Calf-thymus DNA and electrophoretically purified DNAse were purchased from Worthington Biochemical Corp., Freehold, N.J.; and activated DNA was made as previously described (Gerwin and Milstein, 1972). Unlabeled deoxynucleotide triphosphates and dithiothreitol were products of Calbiochem, La Jolla, Calif. Radioactive thymidine triphosphate, methy2t3H3dTTP (20,000 cpm/pmole), was from New England Nuclear Corp., Boston, Mass. Tritiated deoxyguanosine triphosphate 18-3HldGTP (7000 cpmlpmole), was from Schwarz/Mann, Orangeburg, N. Y. Phosphocellulose chromatography. Concentrated virus pellets were resuspended in l-ml disruption solution [0.05 M TrisHCl (pH 7.8), 0.5 M KCl, 1 mM dithiothreitol, 1% Triton X-100, and 20% (by vol) glycerol] and incubated 30 min at 37”. The disrupted sample was centrifuged at 100,000 g for 1 hr at 4”, and the pellet

315

discarded. Supernatant was dialyzed against 100 ml of equilibration buffer for phosphocellulose for two 1-hr periods and applied to a 2.0-ml phosphocellulose column as described previously (Gerwin et al., 1973). DNA polymerase assays. Enzyme activity was determined by assays of lo-p1 aliquots of enzyme preparations in total reaction mixtures of 50 ~1 as described previously (Gerwin et al., 1973; Gerwin and Milstein, 1972). Ingredients of reaction mixtures were as before (Gerwin et al., 1973; Gerwin and Milstein, 1972), except for addition of the indicated primertemplates. Assays for polymerase inhibition by antisera or primer-templates were performed where incorporation was linear with enzyme concentrations. Rabbit antiRD-114 virus polymerase serum (R-antipol(RD-114)) and rabbit anti-MuLV polymerase serum (R-anti-p01 (MuLV)) were prepared as previously detailed (Gilden et al., 1971). Goat immune serum (G-anti-p01 MuLV) directed against Rauscher MuLV polymerase was obtained by immunization with Rauscher MuLV RNA-dependent DNA polymerase partially purified by (dT),,-,,-cellulose chromatography (Gerwin and Milstein, 1972). This serum was kindly supplied by Huntingdon Research Laboratories (Baltimore, Md.). Guinea pig immune serum directed against RD-114 virus RNA-dependent DNA polymerase (GP-anti-pol(RD-114)) was kindly supplied by Dr. Raymond Gilden of Flow Labs, Rockville, Md. Enzymes were preincubated with or without IgG at 4” for 10 min in buffer and then assayed for enzyme activity by addition of template primer and deoxynucleotide triphosphate. Molecular hybridization. Each hybridization reaction was incubated at 66” for varying periods of time as indicated and contained in 0.05 ml: 0.2 M Tris-HCl, pH 7.2, 0.60 M NaCl; 0.05% (v/v) SDS; 5 x lo-” EDTA; 20 pg yeast ribosomal RNA; 5 kg of calf thymus DNA; and approximately 2000 TCA precipitable cpm of [3HlDNA complementary to either RD-114 or M-MuLV. Hybridization was assayed by Sl nuclease method as previously described (Scolnick et al., 1974).

316

PEEBLES, GERWIN AND SCOLNICK RESULTS

Generation and characterization of MSV(RD-114) rescued from S+Lhuman cells. A characteristic of the S+L-

strain of MSV is that it usually permits generation of virus stocks containing MSV in excess of attending helper virus (Bassin et al., 1973; Peebles et al., 1971). Four x lo5 S+L- HuA cells were pretreated with DEAE dextran (25 pg/ml x 60 min), superinfected with RD-114 virus (m.o.i. = 0.051, and carried in tissue culture using methods previously described (Peebles et al., 1973a). Virus of cell-free supernatant culture fluids harvested on Day 28 had titers determined at the indicated dilutions and plotted by the method of Hartley and Rowe (1966; Fig. 1). Titer estimates of MSV(RD-114) were calculated, dilution x FFU/ml, on F-49-l HuA cells. Titer estimates, FIU/ml x dilution, of RD-114 virus were calculated from foci induced on F-1605 human cells. RD-114 virus superinfection of S+L- HuA cells, as described in Fig. 1, rescues transforming MSV with a titer of 104.3FFU/ml while attending RD-114 virus present has a titer of only 102’3FIU/ml. Half-log dilutions of the above indicated virus stock were quantitated for RD-114 virus by the three biological asays outlined in the Methods section. By these methods, it was determined that the FIU virus titer of RD-114 virus present in the stock described in Fig. 1 corresponds to 1020VRU/

ml calculated from induction of RD-114 p30 antigen on human F-49-l cells and lo*” VRU/ml calculated in a two-step assay by detection of focus-forming MSV rescued by RD-114 virus dilutions from superinfected S+L- HuA cells (Table 1). These same assays are able to detect the larger quantities of helper virus released from RD-114 cells. Minimal differences between assays is due most probably to biological variation. These three assay types appear capable of detecting the biologically competent RD-114 virus present in each of the virus stocks. The excess of MSV over biologically detectable RD-114 virus has been further confirmed by the ease with which new S+L- human and dog cells are generated simply by infecting cells with MSV(RD-114) obtained by diluting beyond the end-point titer of RD-114 virus present in the stock. Generation MSV(RD-114)

and characterization rescued from S-+L-

of dog

cells. The maximum titers of MSV (-104) and RD-114 (-lo? rescued from superinfected S+L- human cells are quantitatively insufficient for hybridization studies. To obtain high titer MSV(RD-114) and also to study the origin of MSV polymerase in a heterologous S+L- host cell of another species, S+L- DoCl, cells were in like manner superinfected with RD-114 virus. A 160-ml clarified pool of supernatant culture fluids was harvested at 16 days. TABLE

1

RD-114 VIRUS TITER DETERMINATIONS EMPLOYING DIFFERENT ASSAYS Assay method

RD-114 virus obtained from: RD-114 superinfected S+L- HuA Cl,-, ce119”

1

I

I

IO’

I02

I03

I/DILUTION

FIG. 1. Titration kinetics of MSV(RD-114) (A) and RD-114 (0) virus titers in an MSV stock rescued from RD-114 virus superinfected S+L- human amnion cells.

RD-114 cell&

Focus induction (FIU/ml) Virus rescue WRU/ml) RD-114 p30 induction (VRU/ml) a RD-114 virus present in the MSV (RD-114) virus stock described in Fig. 1. * Virus obtained from filtered supernatant culture fluids of RD-114 cells as described in Methods.

317

MSV DEFECTIVENESS

The rescued MSV(RD-114) titer in this harvest was 2.2 x lo6 FFU/ml on F-49-l HuA cells; and the associated helper RD114 titer was 1.2 x lo6 FIU/ml on S+LMiCl, cells. Eight hundred milliliters of supernatant culture fluid was clarified at 5000 g for 10 min and then pelleted at 100,000g for 2.0 hr, and the RNA was obtained by phenol extraction (Benveniste and Scolnick, 1973; Scolnick et al., 1972). The deproteinized RNA was suspended in 2.0 ml of 0.02 M Tris-HCl, pH 7.0, and 0.010 ml was tested in each hybridization against 3H-labeled cDNA probes of MuLV and RD-114 virus (Fig. 2). The MuLV sequences present in MSV are a measure of its presence (Benveniste and Scolnick, 1973). No sarcoma virus specific probe is as yet available. There is no cross-hybridization between MuLV and RD-114 and their representative DNA transcripts (Benveniste and Todaro, 1973). The results of the hybridization experiments, presented in Fig. 2, show that the amount of MSV RNA in the stock necessary for saturation of MuLV cDNA is equivalent to the amount of RD114 RNA in the same preparation necessary for saturation of the RD-114 cDNA.

.

!I E

0. iI

I lo-'

Rx' loo &.I Irnl I nr*1

I

FIG. 2. Hybridization of MSV(RD-114)/RD-114 RNA to 3H-labeled cDNA of RD-114 virus and MuLV. Deproteinized RNA suspension from MSV(RD-llQ)IRD-114 virus was tested in each hybridization for times ranging from 10 min to 24 hr. V$ [volume (V,) of culture medium from which the RNA was derived multiplied by the time (t, in hr) of hybridization] was calculated as suggested by Reingold et al., 1975. The PH]DNA probe of RD-114 (0) and MuLV (0) hybridized at saturation to approx 1500 TCA cpm, and all values have been normalized to 100% based on this final extent.

The nucleic acid hybridization data thus indicate a l:l, or slightly greater than l:l, ratio of MSV to RD-114 in the virus stock. These titers are quite comparable to the ratio of titers obtained by biological assay of the same stocks, W3 FFU/ml MSV to 106.’ FIU/ml RD-114 virus. Isolation and characterization of DNA the MSV(RD-114) polymerase from stocks. MSWRD-114) virus from human

S+L- cells was rapidly concentrated by ultracentrifugation from 500 ml of clarified culture supernatant of the MSV excess stock described in Fig. 1. The virus pellet was dissolved in 300 ~1 of disruption solution (see Methods); and 5.0 ~1 of this disrupted virus, when assayed for reverse transcriptase using poly(rA)*oligo(dT) by previously described methods, incorpo-

24

i: ' : : 1 t

FIG. 3. Phosphocellulose chromatography of MSV(RD-114) DNA polymerase. Two hundred and fifty milliliters of supernatant culture fluid from the RD-114 virus superinfected S+LHuA cells, described in Fig. 1, were clarified (2x) by centrifugation (2000 g, 20 min, 4”). Virus was concentrated by centrifugation through 20% glycerol for 2 hr at 54,000 g, and the resultant virus pellet was disrupted and applied to a phosphocellulose column, as described in Methods. The column was eluted with a 180-ml linear gradient from 0.1-0.5 M KC1 in equilibration buffer, and 0.01-ml aliquots of the fractions were assayed in 0.05-ml reaction mixtures as previously described (10). (0) Template primer-directed DNA polymerase activity with poly(rA).oligo(dT); (0) DNA-dependent DNA polymerase activity assayed with “activated” calf-thymus DNA (Gerwin et al., 1972); (A) KC1 concentration,

318

PEEBLES,

GERWIN

rated, 509,000 cpmL3HlTMP (20,000 cpm/ pmole) ‘into acid-precipitable material (Gerwin _etal., 1973). The viral polymerase of a 50%,aliquot of the disrupted virus was partially purified chromatographically on phosphocellulose (Fig. 3). MSV (RD-114)/ RD-114 virus from S+ L- dog cells was likewise concentrated from the other 800ml aliquot of clarified culture supernatant of the stock described in the above hybridization experiments. The viral polymerase of this stock was partially purified by phosphocellulose chromatography. The DNA polymerase of the human MSV(RD-114) stock was compared to the DNA polymerase of RD-114 virus and RMuLV for primer-template preference (Table 2). All three of the virion DNA polymerases preferred RNA to DNA templates. All three were most active with poly(A)$dT),,-,B preferring this templateprimer to poly(C)+(dG),,-,, by approximately sixfold in each case.

AND

SCOLNICK

1972; Ross et al., 1971; Scolnick et al., 1972; Scolnick et al., 1972b). The DNA polymerase of RD-114 virus, an endogenous helpertype virus of the feline-primate RD-114/ CCC/M-7 virus group is immunologically distinct from the polymerase of the murine viruses (Fischinger et al., 1973; Scolnick et al., 1972a; Scolnick et al., 1972b). Therefore, antisera may be used to determine the species of origin of the polymerase of MSV rescued from S+ L - human and dog cells by RD-114 virus. The MSV stock (99% MSV and 1% RD114 by biological assays) obtained from S+L- human cells was tested for inhibition by previously described anti-viral polymerase sera (Parks et al., 1972; Ross et al., 1971; Scolnick et al., 1972a; Scolnick et al., 1972b). The partially purified enzyme was found to be inhibited only by rabbit anti-RD-114 virus polymerase serum (Ranti-pol(RD-114)) and not by rabbit antiMuLV serum (R-antipolymerase pol(MuLV)) or control rabbit serum (Fig. Detection of only helper virus polymer4A). To assure that the purification procase in MSV(RD-114) stocks. Antisera against the polymerase of murine type C ess had not selected for only RD-114 virus viruses distinguish these enzymes from polymerase, the polymerase extracted the reverse transcriptase present in nor- from disrupted crude pellet of rapidly conmal cells and from some of the polymer- centrated MSV(RD-114) was tested and ases in other RNA-dependent DNA polym- shown to be inhibited only by R-anti-p01 erase containing viruses (Parks et al., (RD-114) with no significant inhibition by R-anti-p01 (MuLV) (Fig. 4B). To determine whether the presence of TABLE 2 one polymerase interfered with the detecPRIMER-TEMPLATE PREFERENCES OF MSV(RD-114), tion of the other polymerase and to evaluRD-114, AND R-MuLV DNA POLYMERASE ate the sensitivity of these particular antipmole [3HITMP incorporation/ Primer-template sera, reconstruction experiments were perhr/lO A purified formed. Chromatographically MSly4pDRD-114b R-MuLV’ MuLV and RD-114 virus polymerases were individually diluted until their respective 4.0 10.0 1.5 poly(A).(dT),,-,, enzyme activities per unit volume using
319

MSV DEFECTIVENESS

FIG. 4. Inhibition of MSV(RD-114) polymerase and MuLV/RD-114 virus polymerase mixtures by antisera against MuLV and RD-114 virus polymerases. (A) Chromatographically purified polymerase of MSV(RD-114) (A), illustrated in Fig. 3, was assayed for inhibition by control rabbit serum (-. -). R-anti-pol(RD-114) (), and R-antipol(MuLV) (- - -). Five-microliter aliquots of MSV(RD-114) polymerase (initial activity = 28,000 cpm [:‘H]TMP incorporation using poly(rA).oligo(dT) were assayed in 0.06-ml reaction mixtures with the indicated quantities of sera IgG present, as previously described (Parks et al., 1972; Scolnick et al., 1972a; Scolnick et al., 1972b). (B) Polymerase from disrupted crude virus pellet of MSV(RD-114), described in Fig. 1, was assayed as in A above. (C, D) RD-114 virus reverse transcriptase (A) was partially purified by the methods described in Fig. 2, and diluted in buffer until 10 ~1 had -120,000 cpm initial enzyme activity assayed as described with poly(rA)-oligo(dT). Rauscher MuLV polymerase (0) was likewise isolated and diluted until 10 ~1 had -150,000 cpm initial enzyme activity with the same hybrid template. MuLV polymerase and RD-114 virus polymerase were mixed: five parts MuLV pol and tive parts RD-114 pal(0); three parts MuLV pol and seven parts RD-114 pol (0); one part MuLV pol and nine parts RD-114 pol (B). The mixed and unmixed polymerases were assayed for inhibition in C by R-anti-p01 (RD-114) and in D by R-anti-p01 (MuLV).

lymerase present; the antiserum best inhibited the 100% RD-114 polymerase and exhibited the expected decrease in inhibition, as the amount of MuLV polymerase increased. R-anti-pol-MuLV reciprocally and increasingly inhibited reactions of mixtures containing a larger and larger proportion of MuLV polymerase. These reconstruction experiments demonstrate that the presence of either polymerase

fails to inhibit the activity of the other. Moreover, the rabbit antisera are capable of identifying MuLV polymerase when it is 30% or more of the enzyme being assayed. Using separate antisera obtained from goat and guinea pig, the partially purified DNA polymerase from the MSV stock rescued from S + L - dog cells was assayed in like manner for inhibition. GP-antipol(RD-114) again inhibited the enzyme of MSV(RD-114) as if only the RD-114 polymerase were present (Fig. 5A). The inhibition of MSV(RD-114) reverse transcriptase by larger quantitities of G-anti-pol(MuLV1 (Fig. 5A, B) was comparable to that seen with RD-114 viral polymerase alone (Fig. 5B). A reconstruction experiment was again performed to determine the ability of Ganti-pol (MuLV) serum to detect murine polymerase in mixture with RD-114 viral polymerase (Fig. 5B). The results indicated that this antiserum was quite sensitive and able to detect the murine-viral polymerase when present as only lo-20% of the mixture. DISCUSSION

Incorporation of adequate quantities of competent DNA polymerase into virions appears essential for MSV infectivity and transformation, as indicated by the association of polymerase deficiency with the absence of MSV infectivity (Peebles et al., 1972) and the inhibition of MSV focus formation by polymerase inhibitors (rifampitin derivatives and polyadenylic acids) (Tennant et al., 1973; Ting et al., 1972). However, once MSV has transformed a cell it is defective and unable to thereafter replicate infectious progeny in the absence of a replicating helper virus. Previous data have led to the suggestion that certain sarcoma viruses in both the avian and murine systems may in fact be defective because they lack the information for a reverse transcriptase. C-type virus particles released from hamster cells transformed by MSV-Gazdar and from S+L- mouse cells lacked infectivity and have a quantitative deficiency of RNA-dependent DNA polymerase (Bassin et al., 1973; Peebles et al., 1972). Complete ab-

PEEBLES,

GERWIN

AND

SCOLNICK

thus isolated in these nonmurine heterologous host cells failed to express any detectable murine-viral DNA polymerase. However, some murine-viral reverse transcriptase remained associated with MSV expression in the parental S+ L- mouse i,4 cells. These cells released noninfectious vi,,, 25 rus-like particles containing quantitatively I: deficient but detectable amounts of viral E 125 z reverse transcriptase (Bassin et al., 1973) > d 100 easily inhibited by R-anti-pol(MuLV) (70% a with 25 pg IgG) (unpublished data, Pee0.5 75 bles and Gerwin). 50 The possibility that the MSV had been altered by passage into the heterologous 25 host cells was eliminated by back passage of the genome into mouse cells (Peebles et al., 197513).The new S+L- mouse cells again demonstrated release of murine reverse transcriptase into their culture suFIG. 5. Inhibition of MSV(RD-114) virus DNA pernatant fluids. Another hypothesis expolymerase from dog cells and MuLV/RD-114 virus plaining the lack of murine viral reverse polymerase mixtures by antisera against MuLV and RD-114 virus polymerase. (A) Ten-microliter alitranscriptase expression in human, dog, quots of MSV(RD-114) polymerase (initial activity = and mink S+L- cells was that the MSV 150,000 cpm 13H]TMP incorporation using genome coded for the necessary informapoly(rA).(dT),2518)were assayed in 0.05-ml reaction tion, but its expression in those cells is mixtures with the indicated quantities of G-anti-p01 prevented by possible heterologous hostMuLV (-) and GP-anti-pol RD-114 (- - -) cell control (Peebles et al., 197513). present, as described in Methods. (B) RD-114 virus If heterologous host S+L- cell control and RLV reverse transcriptases were partially purisuppresses viral polymerase expression in tied by phosphocellulose chromatography, as dethe absence of replicating helper virus, scribed for MSV(RD-114) virus polymerase (Fig. 3). this suppression should be overcome when RLV polymerase (0) (150,000 cpm 13H1TMP incorporation per 10 ~1) and RD-114 virus polymerase (0) MSV progeny are rescued from those cells. (150,000 cpm [3H1TMP incorporation per 10 ~1) Presuming that all functions coded for by were mixed in the following proportions: nine parts the MSV genome are expressed during resRD-114, one part RLV (A); eight parts RD-114; two cue, murine polymerase should then be parts RLV (A); seven parts RD-114, three parts RLV detected in the infectious MSV virions. (Cl); six parts RD-114, four parts RLV (m); 1 part RDHence, the MSV stock obtained from 114, 1 part RLV (0). These mixtures, the unmixed S +L- human cells and determined by biopolymerases, and MSV (RD-114) virus polymerases logical assays to contain approximately (0) of the same initial activity were assayed for 1OO:l MSV to RD-114 virus, should have inhibition by G-anti-pol(MuLV) (-). only murine polymerase detectable. Fifty to sixty percent of the reverse transcripsence of reverse transcriptase has been tase should in like manner be murine in found in an o-type Rous sarcoma virus the MSV stock obtained from S+L- dog (RSV), a noninfectious variant of RSV in cells. Reconstruction experiments indicate the avian system (Hanafusa et al., 1972; that our serological methods are sufficiently sensitive and capable of detecting Hanafusa and Hanafusa, 1971). We have recently isolated and studied the murine polymerase in mixture with focus-derived cell clones of human, dog, the helper RD-114 virus polymerase (Fig. 4C and D, and Fig. 5B). Yet, only the and mink cells transformed by S+Lstrain of Moloney MSV in the absence of polymerase of the rescuing helper virus detectable helper virus (Papageorge et al., RD-114 is detected in both of these MSV 1974; Peebles et al., 1975b). Defective MSV stocks.

321

MSV DEFECTIVENESS

These results, together with the previous data eliminating the possibility that the MSV genome used in these studies was altered by passage through heterologous host cells (Peebles et al., 1975b), provide strong evidence that the S+Lstrain of MSV is truly defective in genome information for viral reverse transcriptase. If the genome contains this information, then it is not expressed in the absence or presence of replicating helper virus. If the MSV genome contains information for polymerase but it is not expressed in the heterologous host-cell system, then gene regulation might be operating. That regulation would have to selectively control the expression of MSV and not RD-114 virus. Moreover, the murine p30 determinant is present in all heterologous S+Lcells (Peebles et al., 1975b) and also in MSV virions rescued from these cells with RD-114 virus (Peebles, unpublished data). This would indicate that gene regulation does not operate on all MSV genes but is selective for at least the polymerase protein Such selective control, while possible, would appear unlikely. The small amount of murine-viral reverse transcriptase released in noninfectious virions from homologous mouse cells may be an expression of murine helper virus endogenous to the normal 3T3FL cells after interaction or complementation with the S +L- strain of MSV. An endogenous helper virus has, in fact, recently been isolated from these cells (Fischinger and Nomura, 1975). Also, infectious MSV pseudotype has been induced from S+Lmouse cells by halogenated pyrimidine (Nomura et al., Virology, in press). Defective MSV may possibly be studied best by isolation in heterologous host cells lacking the endogenous murine type-C helper viruses (Levy et al., 1974). In addition to the data discussed here, a temperature sensitive mutant (ts29) of Rauscher murine leukemia virus (RMuLV) is defective in a postpenetration function(s) required for replication and has a thermolabile reverse transcriptase (Tronick et al., 1975). Kirsten MSV pseudotypes obtained with this R-MuLV are defective at the nonpermissive temperature for initiation of transformation. This

/defect in the MSV pseudotype indicates that the reverse transcriptase of K-MSV is required for initiation of transformation and also that the enzyme may be supplied to MSV by the ts29 R-MuLV helper virus. The lack of information for reverse transcriptase is apparently not the only replication defect of MSV. MSV rescued from S+Lheterologous host cells is also dependent upon the superinfecting helper virus for its type-specific virus envelope antigen(s) determining host range, neutralization, and interference properties (Hellman et al., 1974; Papageorge et al., 1974; Peebles et al., 1973a; Peebles et al., 1973b; Peebles et al., 1975b). These type-specific envelope moieties may be related to the viral envelope glycoprotein gp71 (Hunsmann et al., 1974). The observations that heterologous host S+L- cells lack any detectable viral-type reverse transcriptase (Peebles et al., 1975b) and that only helper virus polymerase is found in infectious MSV virions (vi& sup-a) are strong evidence arguing that MSV lacks the gene(s) for its own reverse transcriptase. Thus, MSV requires for production of infectious progeny the functioning of a helper virus replication gene set supplying at least viral envelope antigen(s) and the core protein polymerase. ACKNOWLEDGMENTS The authors are grateful for assistance from Mr. Alex Papageorge, Ms. Susan Smith, and Mr. James Fernandez; certain enzyme inhibition assays performed by Mr. Lou Fedele; and the gs-l(p301 antigen determinations performed by Mr. Paul Hill and Dr. Charles J. Sherr. This research was supported in part by a contract of the Virus Cancer Program. REFERENCES AARONSON, S. A., and ROWE, W. P. (19701. Nonproducer clones of murine sarcoma virus transformed BALB/3T3 cells. Virology 42, 9-19. AARONSON, S. A., TODARO, G. J., and SCOLNICK, E. M. (1971). Induction of murine C-type viruses from clonal lines of virus-free BALB/3T3 cells. Science 174, 157-159. BA~NN, R. H., TUTTLE, N., and FISCHINGER, P. J. (1970). Isolation of murine sarcoma virus transformed mouse cells which are negative for leukemia virus from agar suspension cultures. Int. J. Cancer 6, 95-107.

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