Intracellular RNA complementary to the RNA genome of the Moloney-murine sarcoma virus complex

VIROLOGY

100,

288-299

(1980)

Intracellular RNA Complementary to the RNA Genome of the Moloney-Murine Sarcoma Virus Complex JOHN E. KNESEK, MICHAEL A. NASH, JAMES C. CHAN, RICHARD J. BARTLETT, JAMES M. BOWEN, AND JAMES L. EAST’ Department

of Molecular Carcinogenesis and Virology, The University of Texas System Cancer Center, M. D. Anderson Hospital & Tumor Institute, Houston, Texas 77030 Accepted September 11, 1979

Intracellular RNA complementary to genomic RNA of the Moloney-murine sarcoma virus complex was detected in virus-producing rat cells. Hybrids formed between this novel type of RNA and its homologous viral genomic RNA were very stable exhibiting a T, of 88”. Further characterization of the virus-specific complementary RNA revealed that it represented a minimal 74% of the viral RNA genome. Cross-hybridization data of hybrids formed between this viral complementary RNA and normal rat liver RNA or genomic RNA of several other mammalian retroviruses demonstrated its virus specificity and sequence relatedness. An examination of total cellular RNA from virus-producing cells for the number of intracellular virus-specific RNA copies disclosed 14 copies of viral complementary RNA per cell and 1272 copies of viral genomic RNA per cell.

INTRODUCTION

The replication of retroviruses requires that the viral RNA genome be transcribed via an RNA-dependent DNA polymerase into a complementary DNA (cDNA) transcript which subsequently is integrated into the DNA genome of the host cell. These integrated viral nucleic acid sequences then are transcribed back into viral, positivestrand genomic RNA (gRNA) which either is translated into viral proteins on cellular polyribosomes (Fan and Baltimore, 1976; Naso et al., 1973; Shanmugan et al., 1974; Wang et al., 1972) or is incorporated into progeny virions (Brian et al., 1975; Canaani et al., 1973; Cheung et al., 1972; East et al., 1973a,c). In addition to the existence of intracellular viral gRNA within retrovirus-producing cells, there have been reports that certain retrovirus-cell systems also contain intracellular viral, negativestrand complementary RNA (cRNA) whose function is not yet known (Biswal and Benyesh-Melnick, 1969, 1970; Stavnezer et al., 1976). 1 To whom reprint requests should be addressed. 0042~6822/80/020288-12$02.00/0 Copyright 0 1980 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

288

Our interest in retrovirus-specific cRNA came about through the development of quantitative nucleic acid hybridization procedures to detect retrovirus-related nucleic acids in human neoplastic cells. One such technique we have developed recently is the method of competition hybridization in DNA-excess (East et al., 1978, 1979) that is being used to search for intracellular retrovirus-related RNA. In this assay, constant amounts of 3H-labeled viral gRNA and unlabeled viral cDNA in slight excess are annealed with increasing amounts of unlabeled cellular RNA that serves as a competitor. If viral RNA with the same polarity as the RNA genome is present in cellular RNA, it will compete with viral gRNA for hybridization to viral cDNA, and a decrease in hybridization will occur. During the course of development of this competition hybridization assay, we frequently observed with several retrovirus systems that cellular RNA isolated from retrovirus-producing cells was not able to serve as an effective competitor from the standpoint that incomplete competition occurred in the homoiogous competition hybridization assay. In

MOLONEY-MURINE

SARCOMA

VIRUS

contrast, complete competition was obtained when RNA extracted from virions was used as the competitor in the same assay. A possible explanation for this lack of complete competition using cellular RNA as the competitor is that intracellular virus-specific cRNA could be interfering somehow with competition. The reasons for choosing the Moloneymurine sarcoma virus (MSV - M) -cell system were that this is one system where incomplete competition was observed consistently (East et al., 1978), and it is one that has been reported to contain retrovirusspecific, negative-strand cRNA (Biswal and Benyesh-Melnick, 1969,197O). Accordingly, a study was initiated with the objectives of (i) verifying the presence of intracellular, negative-strand MSV-M cRNA within virus-producing cells and (ii) further characterizing intracellular, negative-strand MSV-M cRNA in terms of its thermal stability, its extent of complementarity with the RNA genome of the MSV-M complex, its virus specificity and sequence relatedness with RNA genomes of other mammalian retroviruses, and its copy number per cell. MATERIALS

AND

METHODS

Virus and cells. The MSV-M isolate, which was obtained from the transformed 78Al Wistar rat cell line (Biswal and Benyesh-Melnick, 1969), is a complex consisting of a replication-defective sarcoma virus component and a helper leukemia virus component that provides a gene function(s) essential for the replication of the sarcoma virus component. The average infectivity ratio of leukemia virus to sarcoma virus in this MSV-M isolate is l&l (East et al., 19’73a). Other cell lines and the viruses they produced included the IC-3T3-19 Swiss mouse cell line releasing Moloney-murine leukemia virus (MuLV-M) (Hiraki et al., 1974), a rat kidney cell line producing Kirsten murine sarcoma virus (MSV-K) (Stephenson and Aaronson, 1971), the JLS/V5 mouse spleen-thymus cell line releasing Rauschermurine leukemia virus (MuLV- R) (Syrewicz et al., 1972), and a mouse thymus cell line releasing Gross-mm-me leukemia virus (MuLV-G) (Hartley et aE., 1969). The GR

COMPLEX

COMPLEMENTARY

RNA

2ii9

mouse mammary tumor cell line producing mouse mammary tumor virus (MY&TV-P) (Ringold et al., 1975) and the human rhabdomyosarcoma cell line releasing the endogenous feline retrovirus RD-114 (McAllister et aZ., 1972) also were employed. These cell lines were maintained and passaged as previously described (East et al., 1973a). As a negative control, cells also were obtained from liver tissue of a normal, 3-month-old male Wistar/Furth rat. Preparation of labeled genomic RNAs. Mammalian retrovirus gRNAs from virusproducing cell cultures were labeled with [5-3H]uridine (Amersham) and processed as described in detail elsewhere (East et ai., 1975). The specific activities of the “H-labeled viral gRNAs were assumed to have the same specific activities as total cellular RNA from the 5-day labeled cell cultures. These were calculated to be 4.9 x 105 cpm/pg. For use in hybridization tests, the 3H-labeled viral gRNAs were stored at -20 in water. Synthesis of complementary DNA. The details of the endogenously instructed RNAdependent DNA polymerase reaction and the extraction of unlabeled MSV-M cDNA followed previously described procedures (East et aZ., 1975). Extraction of cellular and viral RNAY. The following procedure was used to obtain total cellular RNAs from trypsinized, washed Wistar rat cells producing the MSV- M complex or from washed cells from liver tissue of the normal Wistar/Furth rat. The cells, which were suspended in TEN buffer (10 mM Tris-hydrochloride, 1 miM EDTA, and 100 m&Z NaCl, pH 7.5) at a concentration of 5 x 10fi cells/ml, were lysed by adding sodium dodecyl sulfate (SDS) to 1% (w/v> and sonicated for 3 min in a Branson Model W-350 sonifier at a 20% duty-cycle setting. The mixture then was extracted sequentially three times with an equal volume of phenol-chloroform-isoamyl alcohol (PCA, l:l:O.Ol) and two times with an equal volume of chloroform-isoamyl alcohol (l:O.Ol). For each extraction, the nucleic acid mixture was shaken vigorously for 10 see, placed in a 60” water bath for 1 min, and removed and shaken again for 10 sec. The heat-shake treatment was repeated,

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KNESEK

and the phases then were separated by centrifugation at 2000 rpm for 5 min at 25”. Diethylpyrocarbonate (Aldrich) was added to the final aqueous phase at a concentration of 0.01% (v/v>, and the nucleic acid was precipitated three times at -20” for 24 hr by the addition of 2 vol of absolute ethanol and 0.1 vol of 10 M LiCl. Each time, the precipitated nucleic acid was collected by centrifugation in a Beckman JA-20 rotor at 12,000 rpm for 30 min at 4”. After the first and second precipitations, the nucleic acid pellet was dissolved in 10 ml of ABS buffer (10 mM sodium acetate and 50 mM sodium chloride, pH 5.0); and after the third precipitation, it was dissolved in TMC buffer (5 mM Tris-hydrochloride, 2.5 mM MgC&, and 1 mM CaCl,, pH 7.4). Next, the sample was treated with 200 pg/ml DNase 1 (Boehringer-Mannheim, grade 1) at 35” for 2.5 hr to remove DNA and then with 500 pg/ml proteinase K (EM Laboratories) at 35” for an additional 16 hr. Finally, the resulting RNA was extracted sequentially three times with PCA, ethanol precipitated, and recovered by centrifugation as before. Unlabeled MSV-M gRNA was isolated from isopycnically banded virions that were suspended in 2 ml of TE buffer (10 mM Tris-hydrochloride and 1 mM EDTA, pH 7.4) and to which was added SDS to 0.5% (w/v). The lysed viral preparation was layered onto a 35-ml linear gradient of 15 to 30% (w/w) sucrose in TE buffer containing 0.1 M LiCl and 0.5% SDS and centrifuged at 15,000 rpm for 16 hr at 27” in a Spinco SW 27 rotor. Fractions of 1.2 ml were taken; and the MSV-M 50-60 S gRNA was pooled, ethanol precipitated, and recovered by centrifugation as before. For use in the hybridization tests, both total cellular RNAs and MSV-M gRNA were stored at -20” in H,O at concentrations of 1 mg/ml and 10 pg/ml, respectively. The concentration of each RNA sample was determined spectrophotometrically, whereby it was assumed that 1 Azso U is equal to 40 pg/ml RNA. All unlabeled RNA preparations had AIBO: Azso ratios of 1.95 or higher. Removal of excess, positive-strand MSV-M gRNA. To remove the excess of intracellular, positive-strand MSV-M gRNA, total cellular RNA from rat cells

ET

AL.

producing the MSV-M complex was selfannealed in 0.24 M sodium phosphate (SP) buffer (132 mM N%HPO, *7Hz0, and 108 mM NaH,PO, * H20, pH 6.8) at a concentration of 15 mg/ml for 72 hr at 70”. The selfannealed RNA sample was diluted with water to bring the molarity of SP buffer to 0.12 M, and 10 pg/ml RNase A and 100 U/ml RNase Tl were added. After incubation at 37” for 1 hr, 1 mg/ml proteinase K was added to the sample which was incubated at 37” for an additional 16 hr. The treated RNA then was extracted sequentially three times with PCA and lyophilized. After lyophilization, the RNA was dissolved in 1.0 ml of water and chromatographed on a Sephadex G-50 column (0.8 x 22 cm). The RNA-containing fractions were ethanol precipitated and recovered by centrifugation as before. For use in hybridization tests, this enriched cellular RNA preparation, representing 24.45% of starting total cellular RNA unless otherwise indicated, was stored at -20” in water at a concentration of 1 mg/ml. In some instances (see Tables 1 and 2), the enriched cellular RNA that was obtained from rat cells producing the MSV-M complex represented only 4.20% of starting total cellular RNA. The reason for this fluctuation in recoveries is not known, but perhaps it was the result of different batches of ribonucleases being used on the two separate occasions. Nevertheless, this RNA which is referred to as the enriched, intracellular MSV-M cRNA preparation in the text was assumed to contain equal proportions of viral, negative-strand cRNA and viral, positive-strand gRNA. Immediately before annealing, this enriched, intracellular MSV-M cRNA preparation (as well as all other cellular RNA preparations) was heated at 100” for 2 min in a boiling water bath and quick cooled in melting ice to insure that the RNA strands were dissociated. Total cellular RNA from liver tissue of the normal Wistar/Furth rat also was self-annealed in this manner, and it represented 19.54% of starting total cellular RNA. Nucleic acid hybridization techniques. Intracellular, negative-strand MSV-M cRNA was detected by a nucleic acid hybridization technique (East et al., 1975) in which a constant amount of 3H-labeled

MOLONEY-MURINE

SARCOMA

VIRUS COMPLEX TABLE

NEGATIVE-STRAND MSV-M

RNA

“Cl1

1

cRNA IN CELLS PRODUCING THE MSV-M

Unlabeled RNA added (Pd

Pretreatment of RNA before annealing

MSV-M cellular RNA” 700, Initial 700, Initial 700, Initial

Self-annealed plus RNase’ RNase at low ionic strength” Alkali digestion’

MSV-M 1 2 13 26

COMPLEMENTARY

COMPLEX

Hybridization with MSV-M [3H]gRNA* (%) 23 6 1

genomic RNA

Yeast RNA 50

None None None None

I 1 2

None

2

1

” The hybridization values shown were obtained by annealing 3H-labeled MSV-M gRNA (2.04 ng, 1603 epm) with the indicated unlabeled RNAs in 0.025 ml of 0.24 M SP buffer at 70” for 72 hr as described under Materials and Methods. * 700 pg of total cellular RNA from rat cells producing the MSV-M complex was used as starting material for each indicated pretreatment. The RNA remaining was used for annealing. p This pretreatment followed the procedure for removal of excess, positive-strand MSV-M gRNA as given under Materials and Methods; 4.20% or 29.4 pg of RNA was recovered and used in the annealing reaction. d Total cellular RNA in water was heated at loo” for 10 min and quick cooled in melting ice. The dissociated RNA then was treated with RNase and subsequently processed without self-annealing according to the procedure for removal of excess, positive-strand MSV-M gRNA as detailed under Materials and Methods. p Total cellular RNA in 0.3 M NaOH was incubated at 37” for 16 hr and then neutralized. The remaining material was ethanol precipitated after adding 100 pg of yeast RNA as carrier.

MSV-M gRNA (2.04 ng, 1003 cpm) and varying amounts of unlabeled RNA from the enriched, intracellular MSV-M cRNA preparation were annealed in 0.025 ml of 0.24 M SP buffer at ‘70” for ‘72 hr. After hybridization, each sample was diluted to 1 ml with 0.12 M SP buffer and divided into 0.5ml aliquots. One aliquot was treated with 10 @g/ml RNase A, and the other served as a control. Both aliquots then were incubated at 37“ for 30 min, and their acidinsoluble radioactivities determined as previously described (East et al., 1973b). Percentage hybridization is defined as counts per minute of the nuclease-treated sample divided by counts per minute of the untreated sample multiplied by 100. Thermal denaturation of hybrid pairs was examined by annealing 3H-labeled MSV- M gRNA (14.28 ng, 7921 cpm) with either unlabeled MSV-M cDNA (3.50 ng) or unlabeled RNA from the enriched, intracellular

MSV-M cRNA preparation (429 pg) in 0.1 ml of 0.24 ikf SP buffer for 43 hr at 70”. Each hybridization mixture then was diluted to 7.2 ml so that the molar&y of the SP buffer was 0.12 M, and the mixtures were divided into LO-ml portions with each portion being heated for 10 min at an appropriate 5” temperature interval ranging from 70 to loo”. Each portion finally was quick cooled in melting ice and divided into 0.5~ml aliquots which were either not treated or treated with RNase A as described above. Thexmal denaturation data are expressed as pereentage of hybrid remaining after RNase A treatment. The number of intracellular, negativestrand MSV-M cRNA copies was measured by a viral gRNA-excess hybridization technique which is analogous to the excess cDNA hybridization method (Berns and Jaenisch, 197s; Heilmann et al., 1977, Panet and Cedar, 1977). In this probe-excess hy-

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bridization procedure, a constant amount of 3H-labeled MSV-M gRNA (2.04 ng, 1003 cpm) in sequence excess was annealed with varying amounts of either unlabeled RNA from the enriched, intracellular MSV-M cRNA preparation or unlabeled total cellular RNA from rat cells producing the MSV-M complex in 0.025 ml of 0.24 M SP buffer at 70” for 72 hr. Hybrid formation was measured by resistance to RNase A as described above. Under these conditions, the final level of hybridization was not allowed to exceed 20%. In this range, a linear relationship exists between the amount of 3H-labeled MSV-M gRNA hybridized and the amount of unlabeled total cellular RNA added; and the slope is directly proportional to the concentration of MSV-M cRNA in total cellular RNA. The slope is defined as percentage hybridization per microgram of total cellular RNA added; and the quantity (percentage hybridization multiplied by micrograms of input 3H-labeled MSV-M gRNA) is equal to micrograms of MSV-M gRNA (or MSV-M cRNA) in hybrid form. Therefore, the concentration of MSV-M cRNA in total cellular RNA is equal to micrograms of MSV-M cRNA per microgram of total cellular RNA. Having computed the percentage of MSV-M cRNA in total cellular RNA, the copy number of this RNA class was calculated from the equation in which copy number is equal to the quantity (micrograms of total RNA per cell multiplied by percentage of MSV-M cRNA in total cellular RNA) divided by micrograms of viral RNA per virion. The amount of total RNA per cell was 8 x 10e6 pg, and the amount of viral RNA per virion was assumed to be 5 x 1O-12pg (Davis and Nayak, 1977; Hayward, 1977; Hayward and Hanafusa, 1973). The intracellular, positive-strand MSVM gRNA copy number was measured by the technique of competition hybridization in slight DNA excess as detailed elsewhere (East et al., 1979). Briefly, constant amounts of 3H-labeled MSV-M gRNA (2.04 ng, 1003 cpm) and a slight excess of unlabeled MSV-M cDNA (5.00 ng) were annealed with increasing amounts of unlabeled competitor RNA (MSV-M gRNA, total cellular RNA from rat cells .producing

ET AL. TABLE HYBRIDIZATION cRNA WITH

2

OF NEGATIVE-STRAND 3H-L~~~~~~ VIRAL

MSV-M gRNAs”

Percentage hybridization 3H-Labeled gRNA added MSV-M MuLV-M MSV-K MuLV-R MuLV-G MMTV-P RD-114

No RNA

Yeast RNA

0 1 1 0 0

1 1 0 0 1

1

1

0

0

with MSV-M cRNA 36 35 18 14 10 2 2

a The hybridization values shown were obtained by annealing the indicated 3H-labeled viral gRNA (2.04 to 2.05 ng, 1003 to 1009 cpm) with either no RNA, yeast RNA (60 pg), or unlabeled RNA (50 pg) from the enriched, intracellular MSV-M cRNA preparation in 0.025 ml of 0.24 1M SP buffer at 70” for 72 hr as described under Materials and Methods. The enriched, intracellular MSV-M cRNA preparation represented 4.20% of starting total cellular RNA from rat cells producing the MSV-M complex.

the MSV-M complex, or yeast RNA) in 0.025 ml of 0.24 M SP buffer at 70” for 72 hr. Hybrid formation was measured ‘with RNase A as described above. Percentage competition is defined as 100% minus (percentage hybridization of sample with competitor RNA divided by percentage hybridization of sample with no competitor RNA). The percentage of MSV-M gRNA in total cellular RNA was calculated from the ratio of the amount of MSV-M gRNA required to achieve 50% competition to the amount of total cellular RNA required to achieve 50% competition (Colcher et al., 1976; East et al., 1979). This value then was used to compute the MSV-M gRNA copy number as described above in which the percentage of MSV-M gRNA in total cellular RNA was substituted for the percentage of MSV-M cRNA in total cellular RNA. RESULTS

Detection of Intracellular

MSV-M

cRNA

As a first step, unlabeled total cellular RNA from rat cells producing the MSV-M

MOLONEY-MURINESARCOMAVIRUSCOMPLEXCOMPLEMENTARYRNA complex was examined for its ability to anneal with 3H-labeled MSV-M gRNA. Such cellular RNA was enriched for possible intracellular, negative-strand MSV-M cRNA by a two-stage process in which total cellular RNA was self-annealed first to form duplex strands and then treated with RNase to remove the excess of unannealed, positive-strand MSV-M gRNA. Before hybridization, this enriched, intracellular MSV-M cRNA preparation was heated in a boiling water bath and quickcooled to dissociate the duplex structures. As illustrated in Table 1, a hybridization value of 23% was obtained when RNA from the enriched, intracellular MSV-M cRNA preparation was annealed with 3Hlabeled MSV-M gRNA. Although this result suggested that virus-producing rat cells contain intracellular MSV-M cRNA, it was necessary to further show that this observed hybridization was due neither to the presence of contaminating proviral DNA in the cellular RNA, nor to intracomplementary base pairing of 3H-labeled MSV-M gRNA, nor to nonspecific annealing with the viral RNA genome. To eliminate the possibility of contaminating MSV-M proviral DNA, total cellular RNA from rat cells producing the MSV-M complex was subjected to two pretreatment procedures prior to hybridization with 3H-labeled MSV-M gRNA. An RNA sample either was first heat-denatured in water to dissociate double-stranded RNA and then treated with RNase to digest both the resulting single-stranded RNA and any single-stranded RNA already present, or it was subjected to alkaline hybrolysis, a treatment that degrades both RNA forms, but not DNA. When such pretreated RNAs were annealed with 3Hlabeled MSV-M gRNA, hybridization values of only 6 and l%, respectively, were noted (Table 1). These results indicated that the observed hybridization was due to RNA and not to contaminating MSV-M proviral DNA. The 5% difference in hybridization observed with the RNA sample pretreated with RNase after heat denaturation at low ionic strength could represent duplex RNA that reassociated. In terms of the observed hybridization

293

being the result of intracomplementary base pairing within the MSV-M RNA genome itself or of nonspecific annealing with the MSV-M RNA genome, OHlabeled MSV-M gRNA was either selfannealed with varying amounts of unlabeled MSV-M gRNA ranging from 1 to 26 pg or annealed with 50 pg of yeast RNA. Hybridization values of only 1 to 2% were obtained in each of these hybridization reactions (Table 1). These data demonstrated that the observed hybridization was due neither to self-annealing of the MSV-M RNA genome, nor to nonspecific annealing with the MSV-M RNA genome. Taken together, the above results indicated that rat cells releasing the MSV-M complex contain intracellular RNA that is complementary in base sequence to the RNA genome of MSV-M. However, the possibility still remained that intracellular, positive-strand MSV-M gRNA was somehow interacting with SH-labeled MSV-M gRNA. Thermal

Stability

of Hybrid

Pairs

To gain insight into this latter possibility, a comparison was made between the thermal stabilities of MSV-M cRNA: [3H]gRNA and MSV-M cDNA:[3HJgRNA hybrid pairs. Hybrids were formed by annealing 3H-labeled MSV-M gRNA either with RNA from the enriched, intracellular MSV-M cRNA preparation or with unlabeled MSV-M cDNA, and then they were dissociated at appropriate temperature intervals ranging from 70 to 100”. As evidenced in Fig. 1, both kinds of hybrid pairs exhibited rather sharp melting profiles. However, the T, value or temperature at the midpoint of transition for the MSV-M cRNA:C3H]gRNA hybrid pair was 88” or 8” higher than the T, value of 80” that was observed with the MSV-M cDNA:[3H]gRNA hybrid pair. This higher T, value for the MSV-M cRNA$‘H]gRNA hybrid pair is further evidence for the existence of intracellular, negative-strand MSV-M cRNA, since viral RNA:RNA hybrids have been shown to have higher T, values (East and Kingsbury, 1971; Kingsbury, 1967) than viral DNA:RNA

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hybrids (East et al., 1975). The likelihood that intracellular, positive-strand MSV-M gRNA is interacting with 3H-labeled MSV-M gRNA appears to be extremely remote. Moreover, Stavnezer et al. (1976) have demonstrated with an avian sarcoma virus that viral gRNA and either 4 and 5 S RNAs from uninfected avian cells or highmolecular-weight RNA from nuclei of uninfected duck cells form hybrids with T, values of less than 60”, whereas the hybrid formed between viral gRNA and intracellular viral cRNA has a higher T, value of 82”. Thus, the high T, value of 88” for the MSV-M cRNA:r3H]gRNA hybrid pair in this study is consistent with the concept that intracellular, negative-strand cRNA from retrovirus-producing cells is viral and not cellular in origin. Extent of Complementarity Spec@icity

and Virus

During this investigation, a critical question relating to the characterization of

Temperature

PC)

FIG. 1. Melting profiles of MSV-M hybrid pairs. Thermal denaturation of hybrids formed between SH-labeled MSV-M gRNA and either unlabeled RNA from the enriched, intracellular MSV-M cRNA preparation (0) or unlabeled MSV-M cDNA (0) was performed as detailed under Materials and Methods. The hybridization values observed at 70” between 3H-labeled MSV-M gRNA and either unlabeled MSV-M cRNA or unlabeled MSV-M cDNA were 29 and 33%, respectively.

ET AL. I

I

I

25

+l

0 Logto

+2 Cellular

+3 RNA

(pg)

FIG. 2. Extent of transcription of MSV-M cRNA. A constant amount of 3H-labeled MSV-M gRNA was annealed with the indicated amounts of unlabeled RNA from either the enriched, intracellular MSV-M cRNA preparation (0) or the enriched, total cellular RNA preparation derived from normal rat liver tissue (0) as described under Materials and Methods.

intracellular, negative-strand MSV-M cRNA was raised. What portion of the viral RNA genome is represented by viral complementary RNA? The answer to this question was obtained by annealing 3Hlabeled MSV-M gRNA with increasing amounts of RNA from the enriched, intracellular MSV-M cRNA preparation. To demonstrate the viral nature of intracellular, negative-strand MSV-M cRNA, 3H-labeled MSV-M gRNA also was annealed with enriched, total cellular RNA from liver tissue of a normal Wistar/ Furth rat. The results are presented in Fig. 2. No hybridization was detected with enriched, total cellular RNA from normal rat liver tissue even at an input of 600 pg. These data indicated that intracellular, negative-strand MSV-M cRNA is indeed viral in origin. In striking contrast, hybridization values continued to increase as the amount of RNA from the enriched, intracellular MSV-M cRNA preparation was increased. A hybridization value of 74% was observed with this RNA at an input of 600 ,ug, the highest level available for testing. This latter result demon-

MOLONEY-MURINESARCOMAVIRUSCOMPLEXCOMPI~EMENTARYRNA strated that intracellular, negative-strand MSV-M cRNA contains nucleotide sequences that are complementary to at least 74%, if not all, of the viral RNA genome. In addition, an examination of the saturation curve obtained with RNA from the enriched, intracellular MSV-M cRNA preparation (Fig. 2). revealed a smooth, monophasic sigmoidal shape spanning 2.4 log units, a fact suggesting no gross disparity between viral RNA sequences transcribed into negative-strand cRNA compared to positive-strand gRNA (Britten and Kohne, 1968). Thus, intracellular, negative-strand MSV-M cRNA appears to be a representative and fairly uniform copy of the viral RNA genome. To gain additional insight into the nature of intracellular, negative-strand MSV-M cRNA, its virus specificity was determined by cross-hybridization of RNA from the enriched intracellular MSV-M cRNA preparation with 3H-labeled gRNAs of several mammalian retroviruses, including MuLVM which is the helper leukemia virus component of the MSV-M complex used in this study. The results of these crosshybridizations are presented in Table 2. All of the 3H-labeled viral gRNAs exhibited 0 to 1% self-annealing and nonspecific annealing values when hybridized by themselves or with yeast RNA. A relatively high hybridization value of 36% was observed again when RNA from the enriched, intracellular MSV-M cRNA preparation was annealed with 3H-labeled MSV-M gRNA. Likewise, a similar hybridization value of 35% was noted when it was annealed with 3H-labeled MuLV-M gRNA. Lower hybridization values of 18, 14, and 10% were detected with hybrids formed between RNA from the enriched, intracellular MSV-M cRNA preparation and “H-labeled gRNAs of MSV-K, MuLV-R, and MuLV-G, respectively. In contrast, limited hybridization values of only 2% were obtained when RNA from the enriched, intracellular MSV-M cRNA preparation was cross-hybridized with 3H-labeled gRNA of MMTV-P or RD-114. Based on these results, the Moloney identity of MSV-M cRNA was established at least in terms of the helper leukemic virus component, and a definite pattern of se-

295

quence relatedness emerged. The crosshybridization data indicated that intracellular, negative-strand MSV-M eRNA shared numerous sequences with the RNA genome of MuLV-M, less with the RNA genomes of other type-C murine retroviruses, and only few with the RNA genomes of the type-B murine retrovirus MMTV-P and the feline endogenous retrovirus RD-114. Copy

Number of Intracellular Specify RNAs

Virus-

A pertinent aspect of characterizing intracellular, negative-strand MSV-M cRNA was the determination of the amount of this novel RNA type in rat cells producing the MSV-M complex. This was accomplished readily using nucleic acid hybridization techniques in a comparative experiment to measure both the amount of intracellular, negative-strand MSV-M cRNA and the amount of intracellular. positive-strand MSV-M gRNA. The copy number of intracellular, negative-strand MSV-M eRNA was determined conveniently using a “H-labeled gRNAexcess hybridization method that is analogous to a 3H-labeled cDNA probe-excess hybridization technique (Berns and Jaenisch, 1976; Heilmann et al., 1977; Panet and Cedar, 1977). Here, advantage was taken of the fact that the enriched, intracellular MSV-M cRNA preparation should contain equal amounts of viral, negativestrand cRNA and viral, positive-strand gRNA in duplex form. Consequently, the concentration of MSV-M cRNA could be deduced from the concentration of MSV-M gRNA. The copy number of intracellular, positive-strand MSV-M gRNA was measured using the technique of competition hybridization in slight DNA-excess (East et al., 1979). With this assay, a direct measurement of the concentration of intracellular, positive-strand MSV-M gRNA could be made from the ratio of the amount of unlabeled MSV-M gRNA required to attain 50% competition to the amount of total cellular RNA required to attain 50% competition (Colcher et al., 1976; East et aZ., 1979). The 3H-labeled gRNA-excess hybridiza-

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tion results, in which 3H-labeled MSV-M gRNA was annealed either with unlabeled RNA from the enriched, intracellular MSV-M cRNA preparation or with unlabeled total cellular RNA from rat cells producing the MSV-M complex, are presented in Fig. 3A. As expected, an increase in the slope of the lines was obtained with RNA from the enriched, intracellular MSV-M cRNA preparation compared to total cellular RNA. This enhancement represented a 6.‘7-fold increase in the slope of the lines from 0.256% hybridization per microgram of total cellular RNA to 1.704% hybridization per microgram of RNA from the enriched, intracellular MSV-M cRNA preparation. Also, the amount of 3H-labeled MSV-M gRNA entering a hybrid form was linearly proportional to the amount of unlabeled cellular RNA added within the range examined. The results of competition hybridization using either unlabeled MSV-M gRNA, unlabeled total cellular RNA from rat cells producing the MSV-M complex, or yeast RNA as the competitor are illustrated in Fig. 3B. As anticipated, similar kinetic curves of competition were observed with MSV-M gRNA and total cellular RNA, and the amount of RNA required to reach 50% competition was lesser with MSV-M gRNA (0.01996 ,ug> than with total cellular RNA (25.11886 pg). No competition was noted using yeast RNA as the competitor. The concentrations of the two virusspecific RNA types within total cellular RNA were computed from the respective hybridization data. For intracellular, negative-strand MSV-M cRNA, its concentration was derived from the slope of the line obtained with RNA from the enriched, intracellular MSV-M cRNA preparation. This calculated concentration of 0.0035% then was adjusted to reflect what it would have been in total cellular RNA by multiplying by 24.45%, the percentage RNA recovered in the enriched, intracellular MSV-M cRNA preparation. This adjusted concentration of intracellular, negativestrand MSV-M cRNA was 0.0009% (Table 3). The concentration of intracellular, positive-strand MSV-M gRNA, which was computed from the ratio of the amounts of

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+3

RNA lpg)

FIG. 3. Measurement of concentrations of intracellular MSV-M RNAs by hybridization. (A) Viral gRNA-excess hybridization of a constant amount of 3H-labeled MSV-M gRNA in excess annealed with the indicated amounts of either unlabeled RNA from the enriched, intracellular MSV-M cRNA preparation (0) or unlabeled total cellular RNA from rat cells producing the MSV-M complex (W). (B) Competition hybridization of constant amounts of 3H-labeled MSV-M gRNA and unlabeled MSV-M cDNA in slight excess annealed with the indicated amounts of either unlabeled MSV-M gRNA (O), unlabeled total cellular RNA from rat cells producing the MSV-M complex (W), or yeast RNA (A). In the absence of competitor RNA, an average hybridization value of 82% was observed with the homologous MSV-M cDNA:MSV-M [3H]gRNA hybrid pair. The procedural details for these hybridization techniques are given under Materials and Methods.

unlabeled MSV-M gRNA and unlabeled total cellular RNA required to attain 50% competition, was 0.0795% (Table 3). Based on these concentrations, a determination of the copy number for each of these intracellular virus-specific RNAs disclosed that total cellular RNA from rat cells producing the MSV-M complex contained 14 copies of intracellular, negative-strand MSV-M cRNA per cell and 1272 copies of intracellular, positive-strand MSV-M gRNA per cell. Thus, negative-strand MSV-M cRNA represented 1.1% of intracellular virus-specific RNA and positive-strand MSV-M gRNA represented 98.9%.

MOLONEY-MURINE

SARCOMA

VIRUS TABLE

COMPLEX

COMPLEMENTARY

RNA

37

3

DISTRIBUTIONOF INTRACELLULAR MSV-M

RNAs

MSV-M RNA tme

Virus-specific RNA” 6)

Viral copies per cellh

Complementary Genomic

o.ooo9 0.0795

14 1272

Distribution (%) 1.1 98.9

I1The percentage of virus-specific RNA was calculated from the data presented in Fig. 3 as detailed under Materials and Methods. ’ The number of viral copies per cell was computed as outlined under Materials and Methods. Each number given in the table is the nearest integer to the calculated value. DISCUSSION

The results of this investigation not only have confirmed the presence of intracellular, negative-strand MSV-M cRNA in rat cells producing the MSV-M complex, but also have provided new information in terms of its thermal stability, its extent of complementarity with the RNA genome of the MSV-M complex, its virus-specificity and pattern of sequence relatedness to other mammalian retroviruses, and its copy number per cell. In this study, we found that the T, value for the MSV-M cRNA:[3H]gRNA hybrid was a relatively high 88”, which may serve as a distinguishing feature for use in characterizing future retrovirus-specific cRNAs. Moreover, we demonstrated that negative-strand MSV-M cRNA contained sequences complementary to at least 74%, if not all, of the viral RNA genome of the MSV-M complex. The cross-hybridization data provided insight into the Moloney specificity of negative-strand MSV-M cRNA in that this novel viral RNA type clearly shared considerable sequence homology with the RNA genome of MuLV-M, which is the helper leukemia virus component of the MSV-M complex used in this paper. Based on a viral subunit RNA analysis of our MSV-M complex, the viral subunit RNA ratio of the leukemia virus component to the sarcoma virus component is only about 2:l (East et al., unpublished observations). Therefore, if the assumption is made that our MSV-M complex is similar to the one previously described from the standpoint that viral gRNA from the sarcoma virus

component shares 70% sequence homology with viral gRNA from the leukemia virus component (Dina and Beemon, 1977; Dina et al., 1976; Hu et al., 1977), then negative-strand MSV-M cRNA also would share sequence homology with the RNA genome of the sarcoma virus component in our MSV-M complex. However, whether negative-strand MSV-M cRNA has sequences shared with the sarcoma-specific region in gRNA from the sarcoma virus component is not known. With regard to the pattern of sequence relatedness observed between negativestrand MSV-M cRNA and RNA genomes of other mammalian retroviruses, the crosshybridization results should be interpreted with some circumspection since only about 40% of the sequences within negativestrand MSV-M cRNA were scored in hybridization tests. Nevertheless, even with this limitation, we found that this portion of the MSV-M cRNA transcript did not share complete sequence homology with gRNAs of other mammalian retroviruses. In other words, this portion of the MSV-M cRNA transcript is not a common region shared in its entirety with RNA genomes of other mammalian retroviruses. Instead, we noted that negative-strand MSV-M cRNA shared moderate sequence homology with gRNAs of other type-C murine retroviruses and only a limited sequence homology with gRNAs of the type-B murine retrovirus MMTV-P and the feline endogenous retrovirus RD-114. These results are in close agreement with a previously published report where retrovirus-specific cDNA transcripts in excess were employed to measure sequence rc-

298

KNESEK

latedness and where the level of hybridization being scored was much higher (East et al., 1975). Our observation that negative-strand MSV-M cRNA represented 1.1% (14 copies per cell) of intracellular virus-specific RNA compared to 98.9% (1272 copies per cell) for positive-strand MSV-M gRNA is consistent with the results of Stavnezer et al. (1976) who examined an avian retroviruscell system. These investigators deduced with the B’77 strain of avian sarcoma virus that the relative ratios of viral negative-strand cRNA to viral positivestrand gRNA were 1:200 in subcellular nuclear RNA and 1:500 in subcellular cytoplasmic RNA. The role that intracellular, negativestrand MSV-M cRNA plays within rat cells producing the MSV-M complex is not yet known. Conceivably, negative-strand MSVM cRNA could represent intermediates in the production of viral subgenomic RNAs which are smaller than viral subunit RNAs (Stavnezer et aZ., 1976). The presence of such subgenomic RNAs has been reported in several retrovirus-cell systems (Brugge et al., 1977; Conley and Velicher, 1978; Parsons et al., 1978; Weiss et al., 1977). Alternatively, negative-strand MSV-M cRNA could represent the replicative form of viral, positive-strand gRNA, thereby providing either an alternate route for replication of the viral RNA genome or a mechanism for amplification of all, or part, of the RNA genome. Negative-strand MSV-M cRNA also could represent a nonfunctional viral RNA that is transcribed occasionally from the parental strand of the integrated proviral DNA, or it could represent other different, unrecognized functional forms. Obviously, further investigations must be done to elucidate the role that intracellular, negative-strand virusspecific cRNA plays in nucleic acid biosynthesis of retroviruses. ACKNOWLEDGMENTS

We thank Marjorie Johnson, Sandra Englert, and Catherine Castenada for providing skilled technical assistance. This investigation was supported in part by Research Grant CA-16781, awarded by the National Cancer Institute, DHEW.

ET AL.

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