Type C viruses of baboons: Isolation from normal cell cultures

Cell, Vol. 2, 55-61,

May 1974,

Copyright@

1974

by MIT

Type C Viruses of Baboons: Isolation from Normal Cell Cultures G. J. Todaro, C. J. Sherr, Ft. E. Benveniste, and M. M. Lieber Viral Leukemia and Lymphoma Branch National Cancer Institute National Institutes of Health Bethesda, Maryland 20014 J. L. Meinick Department of Virology and Epidemiology Baylor College of Medicine Houston, Texas 77025

Summary Four new type C viruses were isolated from putatively virus-negative baboon lung, kidney, and testicular ceils by cocuitivation with several permissive host ceil lines. The baboon type C viruses are infectious for ceils from various mammalian species, but do not replicate in any baboon ceil lines so far tested. These viruses can be distinguished from other major classes of mammalian type C viruses, including previous isolates from primates, but are most closely related to endogenous feline viruses of the RD-114/CCC group. By immunologic criteria, viral host range, and nucleic acid hybridization studies, the baboon type C viruses are highly related to one another and represent a distinct new class of endogenous primate type C viruses. introduction A feline sarcoma virus transformed culture of baboon testicular cells has been found to release a type C virus (M28) (Melnick et al., 1973) with properties distinct from feline leukemia and sarcoma viruses (Todaro, Tevethia, and Melnic, 1973~). This virus isolate, when passaged in human cells, differed antigenically both from feline leukemia and sarcoma viruses (Snyder and Theilen, 1969; Wolfe Table

1. Oriain

Tvoe Animal0

Virus New

of Baboon

C Virus

et al., 1972) and from previously described type C viruses isolated from a woolly monkey fibrosarcoma (Theilen et al., 1971) and a gibbon ape lymphosarcoma (Kawakami et al., 1972) but showed a partial relationship in both the major group specific protein and reverse transcriptase to the RD-114 virus (McAllister et al., 1972), an endogenous virus of domestic cats (Livingston and Todaro, 1973; Fischinger et al., 1973). While previous experiments did not clearly define the species of origin of the M28 virus, recent studies with another type C virus (M7) isolated from normal baboon placental tissue (Benveniste et al., 1974a) suggest that both the M28 and M7 viruses are members of a distinct class of baboon type C viruses. Like the M28 virus, the M7 virus is immunologically related to RD-114, but not to other primate type C isolates (Sherr et al., 1974b). In this report, we describe the isolation of four additional type C viruses obtained from normal baboon kidney, testicular, and lung cell cultures. By immunologic criteria, viral host range, and nucleic acid hybridization studies, these viral isolates are closely related to the M7 and M28 virus isolates. The baboon viruses identified thus far appear to represent a distinct new type C virus group. Results Baboon cell cultures established from different tissues of four animals were employed for the isolation of type C viruses (Table 1). The derivation of the M7 virus has been described in detail elsewhere (Benveniste et al., 1974a). A culture of testicular cells from animal 587 was transformed by feline sarcoma virus and was used previously in the isolation of the M28 virus (Melnick et al., 1973; Todaro et al., 1973c); the untransformed parent culture was utilized in these studies. Cultures of kidney, lung, and testicular cells from animals 8 and 587 were received viably frozen. Both cultures from animal

Isolates Tissue

Cell Culture Initiated

O/maleb

lung

1966

3

O/maleb

kidney

1966

3

Age (years)

! Sex

Passage Level WhenCocultivated

Isolates

BABB-Lg BABB-K

a a

BAB455-K

455

5/female

kidney

1973

3

BAB567-T

587

1.3/male

testis

1971

26

11 /female

placenta

1973

1.3/male

testis (FeSV transformed)

1971

M7 M28

587

OTissues from animals 6. 455, 567 were obtained from Baylor College of Medicine, virus was isolated was obtained from an animal housed at the Southwest Foundation bThis animal was delivered by Caesarian section at term.

Houston. Texas; the placenta for Research and Education,

1 a (filtrates from which San Antonio,

used) the M7 Texas.

Cell 56

Table Baboon Culture

2. Cocuttivation

of Baboon

Cells with

Permissive

Host: Cell Line%

Cell Animal

IdU

Host

Supernatant Reverse cpm x 10-J ‘H-TMP

Cell Line

Weeks Four 59.6 1.5

after

Transcriptase incorporated*

cocultivation Six 967.6 3.0

Testis

507

DBS-FRhL-1 (rhesus lung) FCfPTh (canine thymuse)

Kidney

455

DBS-FRhL-1 FCfPTh

4.4 4.2

221.1 2.8

+

DBS-FRhL-1 FCf2Th

115.1 42.7

731.5 N.T. N.T. N.T.

Kidney Lung

8

+

DBS-FRhL-1 FCfPTh

506.3 125.7

Kidney

a

-I-

DBS-FRhL-1 FCf2Th

1.6 4.1

* Supernatant cpm/flask.

reverse

transcriptaseactivities

in uninfected

cultures

8 were established from biopsies taken in February, 1966 and were stored as frozen stocks of secondary cultures before the discovery of either the woolly monkey (Theilen et al., 1971) gibbon ape (Kawakami et al., 1972) or RD-114 (McAllister et al., 1972) type C viruses. Secondary cultures of baboon kidney cells from animal 455 were established in September 1973. The baboon cultures themselves produced no detectable type C viruses as measured either by a supernatant reverse transcriptase assay or by a radioimmunoassay for the major group specific protein of the RD-114 virus. The BAB587 testicular cells did not contain virus particles as judged by electron microscopy (Melnick et al., 1973). Subconfluent cultures were cocultivated with two mammalian cell lines previously shown to be permissive for replication of the M7 baboon virus. Some cultures were treated for 24 hr with 30 pg/ml 5-iododeoxyuridine (IdU) prior to cocultivation. The permissive host cells used included a continuous line of rhesus monkey lung cells, DBS-FRhL-1 (Wallace et al., 1973) and the dog thymus cell culture FCfPTh (Benveniste et al., 1974a). Cocultivated cultures were serially transferred once each week and were assayed for supernatant reverse transcriptase activity at two week intervals. Type C viruses were first detected in the cocultivated cultures four weeks after initiating the experiment, as indicated by elevated levels of supernatant reverse transcriptase activity (Table 2). By the sixth week, viruses could be detected in at least one cocultivated culture of each baboon cell type. In the absence of the inducer IdU, virus was detected in cultures containing rhesus lung cells, but cultures containing canine thymus cells remained virus negative. Moreover, untreated cultures containing baboon kidney cells from animal 455 were polymerase negative at the fourth week, whereas both parallel IdU-treated cultures were polymerase positive by

of DBS-FRhL-1

and

FCf2Th

Activity

451 .l 1693 cells

are

less

than

5

x

10-J

this time. These experiments therefore show that infectious virus can be recovered from baboon cells by cocultivation alone, but that higher titers of virus appear to be produced after IdU treatment. In parallel experiments, baboon cells were treated with IdU and were subcultured by themselves without the addition of rhesus lung or dog thymus cells. IdU induction did not result in a detectable rise in supernatant reverse transcriptase activity, nor did culture supernatants taken at various times after IdU treatment cause productive infections of any permissive host cell line. The reverse transcriptases produced by both the M28 and M7 viruses have been shown to be inhibited by antisera directed against the polymerase of the RD-114 virus (Todaro et al., 1973c; Sherr et al., 1974b). A similar degree of cross-reaction is also observed with the polymerases produced by the four new baboon isolates. Figure 1A shows the results of an inhibition study in which polymerases from two of the new isolates (BAB8-Lg and BAB587-T) were compared with the M7 and M28 viral enzymes. With 8.5 pg of anti-RD-114 polymerase IgG, there was more than 90% inhibition of the homologous RD-114 enzyme and 60-75% inhibition of the polymerases of the four separate baboon isolates. Identical levels of inhibition have been obtained with the enzymes of the BAB8-K and BAB455-K viruses (not shown). The enzymes from the type C virus isolated from a woolly monkey and from the Rauscher murine leukemia virus, however, were not inhibited by antisera to RD-114 polymerase at these same levels of IgG (Figure 1A). The extent of reverse transcriptase inhibition by the RD-114 antipolymerase sera is independent of the cells in which the baboon viruses are propagated. The same degree of inhibition is obtained with viruses growing in human cells, rhesus monkey cells, dog cells, or bat cells (see below). Further,

Isolation 57

Figure 1. C Viruses

of Baboon

Effect

Type

C Viruses

of Antipolymerase

IgGs on Viral Enzymes

(A) Immune IgG prepared against the polymerase enzymes from: RD-114 (v-v); simian sarcoma (A-A); Rauscher murine leukemia virus (m-m); (O-O); BAB587-T (0-O); BABE-Lg (0-O).

of Type

of RD-114 versus associated virus M7 (A-A); M28

(B) Immune IgG prepared against the polymerase of the gibbon type C virus versus enzymes from: gibbon type C virus (V-V); remainder of symbols identical to those shown in (A).

enzymes partially purified by Sephadex G-100 gel filtration and phosphocellulose chromatography (Ross et al., 1971) are inhibited to the same extent at a given concentration of IgG as the enzymes of viruses sedimented from supernatant fluids of infected cultures. An antiserum prepared against the type C virus isolated from a gibbon ape (Figure IB) fails to inhibit significantly the baboon type C viral enzymes at levels of IgG which almost fully inhibit the homologous viral enzyme; the polymerase of RD-114 is similarly not inhibited. Moreover, antisera prepared against the polymerases of Rauscher murine leukemia virus and feline leukemia virus also do not significantly inhibit the baboon type C viral enzymes at levels of IgG which are 80-90% inhibitory for the homologous viral enzymes (not shown). Figure 2 shows the results of a competitive radioimmunoassay for the major group specific protein of the RD-114 virus. Extracts of cells supporting the replication of the various baboon type C viruses were used as sources of competing antigens. The relative quantities of gs antigen in the cell extracts are reflected by the levels of protein (3 to 10 pg) required to initiate competition, while antigenic similarities between cross-reactive gs proteins are reflected in the slopes of the individual competition curves. An extract of cells producing the RD-114 virus displaced 100% of the ‘*5l-labeled RD-114 gs protein at a level of 175 pg of competing protein. Similar results have been reported previously with other endogenous feline isolates (Livingston and Todaro, 1973; Sherr et al., 1974b). A total of ten separate isolates of endogenous cat type C viruses of the RD-114/CCC group have now been tested (Sherr, et al., unpublished experi-

Figure 2. Competitive Radioimmunoassays Specific Protein of the RD-114 Virus

for the

Major

Group

Competing antigens are extracts of cells supporting the replication of the RD-114 virus, the simian sarcoma associated virus (SSAV), or the various baboon type C isolates. Points on the curves represent averages of 3-6 determinations. Extracts of dog thymus (FCfPTh), rhesus lung (DBS-FRhL-l), rat NRK cells, or human SV80 cells produce less than 10% displacement of the iodinated RD-114 test antigen at the levels of competing protein shown. RD-114/grown in RD cells(r-7); M7/FCf2Th (A-A); M28/SV80 (o-o); BAB455-K/DBS-FRhL-1 (O-O); BABE-Lg/DBS-FRhL-1 (O-O); BABE-KIFCf2Th (A-A); SSAVINRK (m-H).

ments), and each of them competes in an identical manner to RD-114 in the radioimmunoassay. By contrast, each of the baboon type C viruses, whether propagated in dog thymus (FCBTh) or rhesus lung (DBS-FRhL-1) cells, were less effective as competing antigens as shown by the lower slopes of the competition curves, indicating that they contain gs antigens related, but not identical, to the RD-114 gs protein. An extract of cells supporting the replication of the woolly monkey type C virus did not compete in the RD-114 gs assay (Figure 2), although complete competition was easily detected using equivalent amounts of protein in a radioimmunoassay for woolly type C viral gs protein (not shown). Moreover, several other type C viruses, including isolates from pig, cat (FeLV), and mouse (Rauscher virus), fail to compete in the RD114 assay (Sherr et al., 1974b). These results indicate that the new baboon virus isolates, as a class, share immunological determinants with type C viruses of the RD-114/CCC group, whereas other mammalian type C viruses, including isolates from other primates, do not. The viruses isolated from baboon cells are able to replicate in cultured cells derived from several diverse mammalian species (Table 3). No viral replication was observed in cells of the BAB455 kidney line or in BAB8 spleen cell cultures. The fetal cat strain FFcGOWF, which is permissive for replication of endogenous cat viruses of the RD-114/CCC group (Todaro et al., 1973b), was also not permissive for the replication of the baboon isolates. Although the host range of each of the baboon viral

Cell

58

Table Host

3. Host

of Baboon

Type

Cell

Species

Tissue

BAB455-K

Baboon

Kidney

BABI-Sp

Baboon

Spleen

DES-FRhL-I

Rhesus monkey

Lung

A204

\

Range

Human

Rhabdomyosarcoma

C Viruses Virus Replication BAB455-K M7

Table 4. Focus Formation on Baboon Cells Usina Baboon Type C Virus Pseudotypes of Mouse Sarcoma M26

-

-

-

-

-

+

+

+

+

+

+

Species Origin

BABB-K

Baboon

+

+

-

M7

Baboon

+

+

-

M28

Baboon

+

+

-

Mouse

-

+

+

Mouse

-

+

+

Mouse

-

+

+

24

-

-

516-Z

+

+

+

Lung

+

+

+

MLC (BXN) (S-tropic)

Horse

Skin

+

+

N.T.

Mink

Lung

+

+

+

FFcGOWF

Cat

Fetus

FCfPTh

Dog

Thymus

TblLu

Bat

E. Derm MvlLu

isolates shown in Table 1 has not been exhaustively determined, none of the viruses tested so far has been found to replicate in baboon cells, and no differences in host range have been observed between the different isolates. Pseudotypes of the mouse sarcoma virus (MSV) genome have been produced using the baboon viruses as helpers by infection of an MSV nonproducer mink cell line (Henderson, Lieber, and Todaro, 1974). Similarly, MSV pseudotypes with the S-tropic or xenotropic mouse viruses (Levy, 1973; Benveniste et al., 1974b) have been formed by rescue of the sarcoma genome from the MSV nonproducer mink cells. Table 4 shows that both the mouse and baboon virus pseudotypes form foci (titers greater than 103 focus forming units/ml) on the permissive mink cells. However, none of the three baboon viruses are able to infect and produce transformed foci in normal baboon lung cells. The mouse cells, on the other hand, exclude focus formation by the S-tropic mouse type C virus pseudotypes, whereas the baboon cells are readily transformed. The block to virus replication in the homologous system (endogenous baboon virus infection of baboon cells or endogenous mouse virus infection of mouse cells) therefore appears to involve an early step in virus replication. Pseudotypes of MSV with RD-114 which readily produce foci on mink cells and human cells are also unable to transform the BAB8-Lg cells or normal cat embryo cells. To determine the extent of nucleic acid homology between several of the viruses isolated from baboon cells, single-stranded JH-DNA copies of M7 and M28 viral RNAs were prepared and annealed to the cytoplasmic RNAs of cells supporting the replication of baboon, feline, and gibbon type C viruses. An JH-DNA probe, prepared from the M7 virus grown in human A204 cells, hybridized equally well to the cytoplasmic RNAs of cells supporting the replication of the M7, M28, and BAB455-K viruses

Ability to Produce Foci on: Mink Mouse Baboon (BALB/3T3) (MvlLu) (BABB-Lg)

MSV Pseudotype with:

AT-l

(S-tropic)

of

Endosenous Virus(MSV)

AT-124 is a mouse type C virus replicating in human rhabdomyosarcoma cells (RD) (Todaro et al., 1973b). S16-2 (S-tropic) is a mouse virus induced from a subclone of BALB/3T3 by treatment with iododeoxyuridine that replicates in rabbit SIRC cells and other cells from heterologous species (Benveniste et al., 1974b). MLC (BXN) is a type C mouse virus activated from mixed splenocyte cultures (BALB/c x NIH-Swiss) and isolated using SIRC cells (Sherr et al., 1974b). The origin of the three endogenous baboon tvoe C viruses are described in this reoort.

Figure 3. Extracted

Hybridization from Various

of Baboon ‘H-DNA Viral “Probes” Virus-producing Cultur~es

to RNA

(A) A ‘H-DNA product prepared from the M7 virus grown in human rhabdomyosarcoma cells, A204, (1000 countsIGn was added per hybridization reaction aliquot) was hybridized to RNA extracted from cells producing: the baboon viruses M7(o-o), M26 (e-o), and BAB455-K (A-A); the endogenous feline virus, CCC (A-A); and the gibbon type C virus (0-D). The feline and baboon type C viruses were grown in dog thymus cells (FCfLTh), and the gibbon type C virus was grown in an SV40 transformed human cell line

(svao). (B) A ‘H-DNA product prepared from the M28 virus grown in dog thymus cells, FCfPTh. (1000 counts/min was added per hybridization reaction aliquot) was hybridized to RNA extracted from cells producing the baboon, feline, and gibbon type C viruses listed in (A). Symbols are the same as in (A).

(Figure 3A). Similar results were obtained with a probe prepared from the M28 virus grown in canine FCf2Th cells (Figure 38). Almost all of the sequences represented in the two 3H-DNA products are therefore homologous to the viral RNAs from the other baboon isolates tested. Comparable data are obtained whether the viruses replicate in dog thymus cells or in primate cells. The 3H-DNA probes prepared from both the M7 and M28 viruses do not hybridize to cytoplasmic

Isolation 59

of Baboon

Type

C Viruses

RNAs extracted from cells supporting the replication of the gibbon type C virus (Figure 3). Moreover, previous studies using nucleic acid hybridization have shown little or no relationship of the M7 virus to known groups of mammalian type C viruses (Benveniste et al., 1974a). Using both the M7 and M28 SH-DNA probes, a low level of hybridization was detected, however, with the RNA of cells infected with the endogenous cat virus CCC (Figure 3). An ‘HDNA probe prepared from the CCC virus also hybridizes, to a level of approximately 15%, with the DNA of normal baboon cells and with the RNA of cells infected with baboon type C viruses (Benveniste et al., manuscript submitted). These results complement the data obtained from the immunologic studies and show that endogenous cat and baboon viruses are partially related. The viruses isolated from various baboon cells, then, together constitute a distinct class of type C viruses which are related to one another and can be distinguished from the other mammalian type C viruses that have so far been described; of the known type C viruses, however, they are most closely related to those of the RD-114/CCC group. Discussion The results described above indicate that cell lines derived from normal baboon tissues, including testes, placenta, kidney, and lung, release infectious type C viruses that can be readily isolated by cocultivation of the cells with permissive host cell lines. Once isolated, the viruses are infectious for cells derived from a wide variety of mammalian species and replicate to high titer in various primate and nonprimate cells. However, the baboon type C viruses do not replicate in several different baboon cell cultures. The endogenous baboon type C viruses described here therefore share one important property with infectious endogenous type C viruses of other species, in that most cells of the homologous species are able to restrict the replication of exogenously added virus. This property has also been shown for the group E endogenous viruses of normal chickens (Payne, Pani, and Weiss, 1971; Crittenden, Wendel, and Motta, 1973), the Stropic or xenotropic group of mouse type C viruses (Todaro et al., 1973a; Levy, 1973; Aaronson and Stephenson, 1973; Benveniste et al., 1974b), and the RD-114/CCC group of cat type C viruses (Todaro et al., 1973b). Three of the four new baboon viruses described in this report were obtained from cells treated with 5-iododeoxyuridine. However, even in the absence of an inducer, virus could be recovered from cocultivated cultures. Similarly, cocultivation of uninduced baboon placental tissue with permissive host

cells has led to the isolation of the M7 virus (Benveniste et al., 1974a). In parallel experiments using both induced and uninduced BAB455 kidney cells, virus was detected earlier in IdU treated cultures and was isolated in both canine and rhesus host cell lines. In contrast, more time was required before virus could be detected in cocultivated cultures of untreated baboon cells, and only one indicator cell line became virus positive. Thus, IdU treatment appears to facilitate, but is not required for, the recovery of type C viruses from baboon cells. The ready isolation of the various viruses from normal baboon cell lines suggests that viral information is expressed in certain baboon cells. Studies of viral RNA expression in various baboon tissues show marked differences in the transcription of viral information; some tissues, such as placenta and spleen, show relatively high levels of viral specific RNA (Benveniste et al., manuscript submitted). Transcription of endogenous viral type C information has been reported in avian (Hayward and Hanafusa, 1973) and mouse (Benveniste et al., 1973). cells, but the expression of viral specific information in baboon cells is quite extensive and is similar to the levels of expression of endogenous feline viral specific information in certain, apparently “virusfree” cat tissues and cell cultures (Okabe, Gilden, and Hatanaka, 1973). The nucleic acid hybridization experiments indicate that, as a class, the baboon viruses show a high degree of relatedness to one another, greater than that observed, for example, between different endogenous type C viruses isolated from subclones of the mouse cell line BALB/3T3 (Benveniste et al., 1974b). A limitation on the interpretation of these hybridization data rests on the possibility that some sequences in baboon viral RNA are not transcribed into 3H-DNA. However, single-stranded viral DNA transcripts prepared using actinomycin D are more representative of the entire viral RNA genome than DNA prepared in the absence of actinomycin D (Taylor et al., 1973). Viral specific DNA has been found in different tissues from several animals of different colonies, suggesting that such viral sequences are widespread among baboons (Benveniste et al., manuscript submitted). By contrast, type C viruses previously isolated from tissues of a woolly monkey and gibbon ape do not appear to be endogenous in these species, since DNA sequences homologous to woolly monkey and gibbon ape viral genes cannot be detected in the tissues of woolly monkeys, gibbon apes, or any other primate so far tested (Scolnick et al., 1974). The baboon type C viruses show a partial immunologic relationship in both their reverse transcriptases and major group specific proteins to endogenous feline viruses of the RD-114ICCC group,

Cell 60

but not to other type C viruses, including those previously isolated from primates. The viral proteins of endogenous feline and baboon viruses therefore share certain interspecies determinants not found in other type C viruses, and the antisera used in these studies detect these determinants preferentially. The baboon and endogenous feline viruses are more readily distinguished from one another by nucleic acid hybridization studies, where the degree of homology between the viral classes using the DNA probe from one group and the viral RNA from another is lo-20%. However, even these levels of cross-species hybridization between different endogenous type C viruses from different species are rarely detected. DNA sequences homologous to baboon viral genes have been found in other Old World monkeys, including patas, rhesus, and African green monkeys (Benveniste et al., manuscript submitted). The presence of this viral specific information in these species suggests that a virus related to baboon type C viruses has been present in the genomes of Old World monkeys for the entire time of their evolutionary divergence, estimated at 30 million years (Romero-Herrera et al., 1973). However, the homology between endogenous feline and baboon type C viruses raises the possibility that an infectious virus may have been transmitted from cats to monkeys, or vice versa, and became part of the DNA of the infected species. Experimental

Procedures

Cells and Culture Conditions The baboon cells used in these studies are indicated in Table 1. Cell lines used in cocultivation experiments (Table 2) or viral host range studies (Table 3) included: rhesus monkey lung cells, DBSFRhL-1, a gift from Dr. Roslyn Wallace (Wallace et al., 1973); the human rhabdomyosarcoma, A204, derived in this laboratory (Giard et al., 1973); the bat lung line, TblLu, the mink lung line, MvlLu, and the equine dermal culture, E. Derm, from the American Type Culture Collection; and the Naval Biomedical Research Laboratory lines FFcGOWF (feline fetus) and FCf2Th (canine thymus). All cell lines were grown in Dulbecco’s modification of Eagle’s medium with 10% calf serum (Colorado Serum Company) and were serially transferred using 0.1% trypsin in phosphate-buffered saline. Cocultivation of baboon cells with permissive host cell lines was performed as previously described (Todaro et al., 1973b; Benveniste et al., 1974a). Supernatant Reverse Transcrlptase Assay Concentration of putative virus from the supernatants of cell cultures and assays for viral polymerases were performed as previously described (Todaro et al., 1973b). Poly rA was used as template and oligo dTll.,r as primer in all reactions. Results are expressed as CPM of “H-TMP (40,000 CPM/pmole) incorporated into poly dT product in a 60 min reaction at 37°C. Polymerase lnhlbltlon Studles Rabbit antisera to purified viral polymerases was prepared as described previously (Parks et al., 1972; Scolnick et al., 1972b). Enzyme reactions were initiated with a mixture of template, primer, and substrate; and enzyme was added prior to the addition of anti-polymerase IgG. Dilutions of enzyme were used to yield

50,000-120,000 enzyme.

CPM

of ‘H-TMP

incorporated

for the uninhibited

Radloimmunoassay for Group Specific Protein Competitive radioimmunoassays were performed as described (Scolnick et al., 1972a; Parks and Scolnick, 1972). using antisera prepared against the major gs protein from the endogenous cat virus, RD-114. Assays were initiated using a dilution of antiserum which bound 40-50% of the iodinated test antigen. Under these conditions, the major group specific proteins of Rauscher-MuLV, Rickard-FeLV, the porcine type C virus PK-15, the woolly monkey type C virus, and the gibbon ape type C virus do not compete with the labeled RD-114 gs protein (Sherr et al., 1974b). Competing antigens were prepared by ether extraction of cells producing virus. Suspensions of virus-infected cells in 0.5 ml of tissue culture medium were extracted with 10 volumes of ether, the ether evaporated, and the suspensions clarified by centrifugation at 3000 x g for 15 min before use. Immune complexes were detected by coprecipitation with a titered excess of goat anti-7S immunoglobulin serum (Meloy Laboratories, Springfield, Virginia). Protein in the competing antigens was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Nucleic Acid Hybrldlzatlon A 3 hr endogenous reverse transcriptase reaction from detergentdisrupted type C virus was used to snythesize single-stranded IHthymidine labeled DNA in the presence of actinomycin D (30~~q/ml) as described in Benveniste and Scolnick (1973) Benveniste et al. (1973). Cytoplasmic RNA was extracted from various virus-producing cell cultures as described in Benveniste et al. (1973). Cellular RNA ‘H-DNA hybridizations were incubated at 50°C in reaction mixtures containing 0.01 M Tris, pH 7.4, 0.40 M NaCI. 2 x 10-r M EDTA, 0.05% SDS, 50% formamide, 20,000 CPM/ml of “H-DNA and 2 mg/ml of cytoplasmic RNA. The ratio of the concentrations of RNA to ‘H-DNA was 2 X 106 in these experiments. Hybridizations were started by heating the mixtures to 90°C for 10 min, cooling on ice, and incubating at 50°C. At varying times (ranging from 6 min to 72 hr), 0.05 ml aliquots was removed and frozen at -80°C. The extent of hybrid formation was determined with the single-strand specific nuclease, Sl, as previously described in Benveniste and Scolnick (1973). Duplicate zero time samples were also withdrawn to monitor any effect of cytoplasmic RNA on the hydrolysis of single-stranded )H-DNA product by Sl nuclease. The amount of ‘H-DNA that was Sl resistant in the presence of RNA varied from 3-7%. C,t values (Leong et al., 1972; Birnstiel. Sells, and Purdom, 1972)-RNA concentration x time of incubation-were calculated as suggested by Britten and Kohne (1968) and corrected to a monovalent cation concentration of 0.18 M (Britten and Smith, 1970). Acknowledgment We thank L. Fedele, E. Meyer, and G. L. Wilson search was supported in Program of the National

V. Harvey, R. Heinemann. L. Lahey, C. for assistance in these studies. This repart by a contract from the Virus Cancer Institutes of Health.

Received

January

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