lnteractiqn of psoralen derivatives with the rna genome · lnteractiqn of psoralen derivatives with...

VIROLOGY 113, 613-622 (1981)

lnteractiqn of Psoralen Derivatives with the RNA Genome of Rous Sarcoma Virus

RONALD SWANSTROM,*,’ LESLEY M. HALLICK,?” JEAN JACKSON,* JOHN E. HEARST,? AND J. MICHAEL BISHOP*

*Department of Microbiobgy and Immunology, University of California, San Francisco, Calzfornia 94149, and tDepartment of Chemistry,

University of Cal@rnia, Berkeley, Cal(f&rnia 94720

Received December 17, 1980; accepted April 16, 1981

We have examined the interaction of several psoralen derivatives with the RNA genome of Rous sarcoma virus (RSV). In the presence of long-wavelength uv light, 4,5’,8-trimethylpsoralen (trioxsalen) and its 4’-hydroxymethyl (HMT) and 4’-aminomethyl (AMT) derivatives all efficiently crosslinked the subunits of the RSV 70 S RNA complex into a nondissociable state. These psoralen derivatives also were able to penetrate virions and crosslink the RNA complex in situ. The ability of psoralen to crosslink the subunits of viral RNA in situ is consistent with the presumption that the RSV genome exists as a complex within the virion and suggests an approach for a detailed examination of regions of secondary structure. The addition of psoralen adducts to viral RNA correlated with a loss of virus infectivity and a decrease in the amount and length of viral DNA synthesized in vitro and in vivo. Our results indicate that the RSV genome contains a significant amount of secondary structure that is reactive with psoralen compounds and that the addition of HMT to viral RNA causes premature chain termination during viral DNA synthesis.

INTRODUCTION The utility of these compounds as probes

The psoralens comprise a family of het- for regions of secondary structure has

erocyclic compounds which can, in the been demonstrated for several single- stranded heteropolymers. Single-stranded

Presence Of low-wavelength UV light, SV49 DNA (S&n and Hearst, lg77), fd chemically crosslink the opposite strands of either duplex DNA or RNA (Cole, 1970;

phage DNA (Shen and Hearst, 1976), 18

Dall’Acqua et aZ., 1970; Cole, 1971; Isaacs S and 26 S rRNA from Drosophila mela-

et CLZ., 1977). Due to this property the psor- nogctster (Wollenzien et ak, 1978), and 16

alens offer a probe for exploring regions S rRNA from Escherichia coli (Wollenzien

of secondary structure in single-stranded et aZ., 1979) were each shown by electron

nucleic acids. In addition, several of the microscopy to have regions of secondary

psoralen derivatives Can penetrate living structure identifiable after photoreaction

cells and intact virions, allowing the as- with psoralen derivatives.

sessment of secondary structure in situ We have used several psoralen deriva-

(Pathak and Kramer, 1969; Cole, 1970; t* Ives to study the structure of the RNA

Hanson et al., 1976; Hearst and Thiry, genome of Rous sarcoma virus (RSV), an

1977; Hallick et cd., 1978). oncogenic retrovirus of chickens. The viral genome is a complex of identical subunits held together by intermolecular base-pair-

’ To whom reprint requests should be sent. ing (Coffin, 1979). The subunits are about ’ Present address: Department of Microbiology, 9 kbases in length with a sedimentation

University of Oregon Health Sciences Center, Port- coefficient of 38 S (Duesberg, 1968). The land, Oregon 9’7201. genome complex has a sedimentation coef-

613 0042-6822/81/120613-10$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

614 SWANSTROM ET AL.

ficient of ‘70 S. It is believed that the RSV genome is a dimer (Mange1 et ah, 1974; King, 1976), having a structure analogous to that of the mammalian C-type viruses (Kung et aZ., 19’75; Bender and Davidson, 1976; Kung et aZ., 1976). In this study we show that (i) several psoralen derivatives are able to crosslink the RSV 70 S RNA complex into a nondissociable state; (ii) reaction with at least one of the derivatives decreases viral infectivity; and (iii) the decrease in infectivity is probably a result of inhibition of viral DNA synthesis.

MATERIALS AND METHODS

Cells and viruses. The propagation of B77 strain (subgroup C) of RSV on chicken embryo fibroblasts (CEF) was carried out as previously described (Dahlberg et aZ., 1974). A clonal isolate of the Prague strain (subgroup A) of RSV was obtained from Dr. P. Vogt. Prague strain (subgroup C) of RSV was obtained as concentrated sus- pensions from University Laboratories, Highland Park, New Jersey, under the auspices of the Office of Program Re- sources and Logistics, National Cancer In- stitute. The titer of focus-forming units in virus stocks was determined as described by Vogt (1969).

Preparation and analysis of radioactive viral RNA. The labeling of virus-produc- ing cells with [32P]orthophosphate, puri- fication of radioactively labeled virus, and isolation of viral 70 S RNA were all done as previously described (Bishop et al., 1970; Cordell et aZ., 1979). Thermal denaturation of viral RNA was accomplished by diluting the RNA into a solution of 0.01 M Tris- HCl, pH 7.4, 0.01 M EDTA, pH 7.0, 0.5% SDS, and heating at 80°C for 3 min. Anal- ysis of native and denatured viral RNA by velocity sedimentation was done by cen- trifugation into a 15 to 30% (w/w) sucrose gradient in 0.1 M NaCl, 0.01 M EDTA, 0.01 M Tris-HCl, pH 7.4, 0.5% SDS. Gradients were spun in an SW27 rotor for 14 hr at 18,000 rpm, 21°C. The amount of radioactivity in each fraction was measured as Cerenkov radiation.

Photoreackn of viral RNA with psor-

alen derivatives. The syntheses of the 4’-hydroxymethyl (HMT) and the 4’-aminomethyl (AMT) derivatives of 4,5’,8- trimethylpsoralen (trioxsalen) have been described (Isaacs et al., 1977). Unless oth- erwise indicated, trioxsalen and HMT were used at saturating concentrations (0.5 and 25 pg/ml, respectively) and AMT was used at 25 pg/ml. Irradiation of samples was done at 4°C using two 15-W General Elec- tric F15T8 BLB fluorescent tubes (Shen and Hearst, 1976). In some experiments a concentrated solution of the psoralen de- rivative in DMSO was diluted into virus- containing medium (medium 199 with 1% calf serum). The virus solution was then placed in a plastic petri dish and exposed to the light source from the underside. A 3-mm-thick Plexiglas sheet was placed under the petri dish to shield short-wavelength uv light emitted by the lamps. Treatment of purified virus or RNA was done by diluting the sample at least five- fold into a solution containing 0.02 MTris- HCl, pH 7.4, 0.01 M NaCl, 0.001 M EDTA, and either trioxsalen, HMT, or AMT at the indicated concentration. Irradiation of the sample was most conveniently done in a loo-p1 glass capillary pipet (Drummond) at a distance of 1 to 2 cm from the lamp.

Viral DNA synthesis in vitro. Endoge- nous reaction: Concentrated virus was diluted into a solution containing 0.05 M Tris-HCl, pH 8.1, 0.007 M MgC12, 0.06 M NaCl, 0.01 M DTT, 0.05% NP40 (Shell), 50 fi each dATP, dCTP, and dGTP, and either 1 ti[3H]dTTP (50 Ci/mmol; Schwa& Mann) or 0.5 PM [a(-32P)]dTTP (400 Ci/ mmol; New England Nuclear). Samples were incubated at 37°C for 1 to 2 hr, at which time an aliquot was removed and

incorporation of the radioactive precursor was determined by TCA precipitation and liquid scintillation counting. Radioactive DNA products were isolated by phenol extraction, ethanol precipitation of nucleic acids, and treatment of the precipitates with 0.3 N NaOH to remove the tRNA primer from viral DNA. Alkaline hydro- lysis of RNA was done at room tempera- ture for 12 hr. The solution was neutral- ized with acetic acid and the DNA precipitated with ethanol. The DNA was

PSORALEN DERIVATIVES AND THE RNA GENOME OF RSV 615

resuspended in an aqueous solution containing 50% formamide, heated to 100°C for 3 min, and analyzed by gel electrophoresis in a 5% acrylamide gel containing 8 M urea (Maxam and Gilbert, 1980). The DNA products were visualized by autoradiography using Kodak X-Omat R film and DuPont X-ray intensifying screens (Swanstrom and Shank, 1978).

Exogenous reaction: Poly(rA) and oligo(dT) were added to detergent-dis- rupted virions in the presence of [3H]dTTP as described by Tereba and Murti (1977). DNA polymerase activity was measured by incorporation of the radioactive precursor as measured by TCA precipitation.

Analysis of viral DNA synthesized in viva. Virus-containing medium was treated with HMT and long-wavelength uv light. The virus was then placed on cultures of chicken embryo fibroblasts for 1 hr. At the end of 1 hr fresh medium was placed on the cells. Seven hours later the cells were harvested and unintegrated DNA was isolated by the method of Hirt (1967) as previously described (Shank et aZ., 1978). The fractionation of viral DNA by agarose gel electrophoresis and the detection of viral DNA by hybridization after transfer out of the gel onto nitrocellulose paper were done following published procedures (Southern, 1975; Shank et al., 1978). Viral DNA was detected by annealing the filter with a =P-labeled DNA probe complemen- tary to viral genome RNA, followed by autoradiography (Shank et al, 1978; Swanstrom and Shank, 1978).

RESULTS

Effect of Psoralen on RSV Genome RNA

The RSV genome probably consists of two 38 S RNA subunits held together by intermolecular base-pairing in a 70 S complex (Coffin, 1979). Using various psoralen derivatives we attempted to crosslink the subunits of the genome into a nondissociable form. Radioactively labeled viral 70 S RNA was purified from virions and exposed to 4’-hydroxymethyltrioxsalen (HMT) in the presence of long-wavelength uv light. The viral RNA was then heated

to denature the 70 S complex into the com- ponent subunits and analyzed by velocity sedimentation. Figure 1A illustrates an analysis of unreacted RNA before and after thermal denaturation; the native RNA sedimented as a 70 S complex, whereas the denatured RNA sedimented as a broad peak around 20 to 25 S. Since intact subunit RNA has a sedimentation coefficient of 38 S (Duesberg, 1968), each subunit RNA molecule used in this experiment contained an average of two to four covalent breaks. Figure 1B shows a similar analysis of viral RNA that had been photoreacted with HMT prior to thermal denaturation and sizing. After the addition

I I 1 10 20

Fraction Number

FIG. 1. Crosslinking of RSV 70 S RNA with 4’-hydroxymethyltrioxsalen. Radioactive viral RNA purified from virions was analyzed by velocity sedimentation in a sucrose gradient as described in Materials and Methods. Chicken ribosomal RNA was run in parallel as a marker. The RNA was analyzed in its native state (0 - 0) or after denaturation at 80°C for 3 min (O - 0). (A) Untreated RNA. (B) RNA photoreacted with HMT prior to denaturation and gradient analysis. Sedimentation is from right to left.


of HMT to viral RNA the 70 S RNA complex was in a nondissociable state, i.e., it sedimented as a 70 S structure after thermal denaturation. This effect was observed only when the RNA was exposed to both HMT and uv light; either treatment alone had no effect. We also examined the reactivity of several other psoralen derivatives with the 70 S RNA complex. Both the trioxsalen and the AMT derivatives stabilized the 70 S RNA complex in the presence of uv light (data not shown).

Crosslinking Virion RNA in Situ

Psoralen compounds are able to penetrate whole cells and crosslink DNA within cellular or viral chromatin in situ (Hanson et al., 1976; Wiesehahn et al., 1977; Hallick et al., 1978). The reactivity of psoralen appears to be specific for nucleic acids; these compounds have not. been demonstrated to crosslink proteins or proteins to nucleic acids (Frederiksen and Hearst, 1979). We therefore attempted to crosslink the RSV RNA complex in situ by photoreacting intact virions with HMT. Virus harvested from cells labeled with 32P was photoreacted with HMT. Virion RNA was purified and analyzed by velocity sedimentation either with or without prior thermal denaturation. As shown in Fig. 2A, RNA extracted from virions was dissociated into smaller species after heating. Figure 2B shows the sedimentation pattern of virion RNA after photoreaction with HMT and thermal denaturation. It can be seen that the 70 S RNA complex was essentially unchanged after heating, indicating that HMT penetrated the virion and crosslinked the RNA complex in situ. The two other psoralen compounds gave similar results, although trioxsalen was less ef- ficient in the yield of the stabilized 70 S complex, presumably because of a low- er initial drug concentration (data not shown).

RSV Loses Iqfectivity ajler Treatment with HMT

The mechanism of crosslinking with psoralen involves the formation of chemical bonds between a single psoralen mol-

FIG. 2. Crosslinking of RSV RNA in situ. Virus particles labeled with “P were prepared and photoreacted with HMT. RNA was then isolated from the virions and analyzed by velocity sedimentation in a sucrose gradient as described in Materials and Methods. Chicken ribosomal RNA was run in parallel as a marker. The RNA was analyzed in its native state (0 - 0) or after thermal denaturation (0 -0). (A) RNA from untreated virions. (B) RNA from virions that had been photoreacted with HMT. Sedimentation is from right to left.

ecule and both strands of a double helix (Dall’Acqua et al., 1970; Cole, 1971; Dall’Acqua et aZ., 1972). We examined the possibility that such an alteration of the viral genome would reduce infectivity. Vi- rus infectivity was measured by focus formation on chicken embryo fibroblasts (Vogt, 1969). Figure 3 shows the results of focus assays done on samples of virus photoreacted with HMT for increasing periods of time. From these results it can be seen that HMT, in the presence of long-wavelength uv light, inactivated the focus- forming ability of the virus. The titer decreased by a factor of lo4 in a 30-min exposure at the highest HMT concentration (25 pg/ml).

Loss of Infectivity Correlates with a De- crease in Viral DNA Synthesis in Vi- tro and in Viva

One posible mechanism by which HMT could block virus replication is by cova-


0, \ .E .E \ ? \ 3 m IO-4 - \

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1 I I I I 20 40

Time of Irradiation (min)

FIG. 3. Inactivation of RSV infectivity by HMT. HMT was added at the indicated concentrations to medium containing the B7’7 strain of RSV. The virus was then exposed to long-wavelength uv light. Ali- quots were removed at the indicated times. The aliquots were titered for focus-forming units as described by Vogt (1969). (m - - n ) 0.25 rg/ml HMT, (A - A) 2.5 rg/ml HMT; (0 - - 0) 25 pg/ml HMT, (0 - 0) either uv light alone or HMT at 25 I.cg/ml in the absence of uv light. The initial titer was 1.2 X lo6 focus-forming units/ml.

lently attaching to the RNA template and terminating DNA synthesis. In order to test this hypothesis we compared viral DNA synthesis to the focus-forming titer using virions that had been photoreacted with HMT. We examined both the amount and the size of DNA synthesized in vitro and in vivo.

RSV was treated with HMT and exposed to long-wavelength uv light for increasing periods of time. The treated virus was di- vided into two parts; one part was assayed for the focus-forming titer and the other part was used to synthesize viral DNA in vitro. DNA synthesis was measured as the amount of a radioactive precursor incor- porated into DNA after disrupting the virions with a nonionic detergent (endogenous reaction). The results of these two assays are shown in Fig. 4. As infectivity decreased with time of exposure to long-

wavelength uv light in the presence of HMT, there was also a significant decrease in the amount of DNA that could be synthesized using the treated virions.

The decrease in DNA synthesis after photoreaction with HMT was attributed to modification of the endogenous viral RNA template based on the results of three experiments:

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E 10 B

s .- t e

Y

10

I I 30 60

Time of Irradiation (min)

FIG. 4. Correlation between virus inactivation and a decrease in viral DNA synthesis. Medium containing the B7’7 strain of RSV was made 25 rg/ml with HMT. The virus was then exposed to long-wavelength uv light. Aliquots were removed at the indicated times and titered (0 - 0) as described in the leg- end to Fig. 3. A fraction of each aliquot was tested for DNA synthesis using an exogenous homopolymer template (0 - 0). Another fraction of each aliquot was tested for DNA synthesis using the endogenous viral RNA template. Virus was pelleted from the medium and an endogenous reaction carried out as described in Materials and Methods (A - - A). The initial titer was 7.5 X 105 focus-forming units/ml. The maximum incorporation for the exogenous reaction was 63,299 cpm; the maximum incorporation for the endogenous reaction was 303,334 cpm. Treat- ment with either HMT or long-wavelength uv light alone had virtually no effect on viral DNA synthesis.


123456

FIG. 5. Viral DNA products synthesized using HMT-treated virions. Purified virions (Prague C RSV) were photoreacted with HMT for the indicated periods of time. The virions were then used to synthesize DNA in the endogenous reaction as described in Materials and Methods. At the end of the reaction, DNA was purified and analyzed by polyacrylamide gel electrophoresis. Total incorporation decreased sevenfold at the highest HMT dose compared to the control; the same amount of radioactivity was loaded onto each lane of the gel. SV40 DNA cleaved with the restriction endonuclease Hind111 and end-labeled was run denatured in an adjacent lane as a size stan- dard. Migrating at a position 101 nucleotides in length is the prominent viral DNA product synthesized in vitro, “strong stop DNA” (Shine et al., 19’77; Haseltine et aZ., 1977). Lane 1, long-wavelength uv light alone; lane 2, HMT in the absence of long-wavelength uv light; lanes 3-5, HMT plus uv light for 15 min. 30 min, and 1 hr. respectively. One sample, shown in lane 6, was irradiated for 30 min in the presence of HMT, at which time another dose of HMT was added and irradiation continued for another 30 min.

(i) The virion DNA polymerase was not significantly affected by photoreaction with HMT. The same virions in which endogenous DNA synthesis (using the resi- dent RNA template) was inhibited by up to 90% retained polymerase activity near the level of the control when the poly-

merase activity was measured using an exogenous homopolymer template (Fig. 4).

(ii) RNA extracted from virions photoreacted with HMT supported reduced levels of DNA synthesis in a reaction containing purified polymerase (data not shown).

(iii) Truncated DNA products were synthesized in virions treated with HMT. Pu- rified virions were photoreacted with HMT. These virions were then used to synthesize DNA in the endogenous reaction in the presence of a radioactive precursor. The DNA products were isolated and analyzed by polyacrylamide gel electrophoresis. The gel system was capable of resolving DNA fragments 50 to 500 nucleotides in length. Lanes 1 and 2 in Fig. 5 show DNA products synthesized in virions that had been treated with either long-wavelength uv light or HMT alone. The pattern of premature termination products is identical to that observed in the absence of any treatment (not shown). When virions that had been treated with HMT and long- wavelength uv light together were used to synthesize DNA, a pattern of truncated DNA products was observed (Fig. 5, lanes 3-6). Over 10 new DNA species were synthesized using a template that had been photoreacted with HMT.

We have also determined the effect of HMT treatment of virions on viral DNA synthesis in viva. A virus stock was photoreacted with HMT for increasing periods of time. This stock was then titered for focus-forming activity and used to infect chicken embryo fibroblasts. At 8 hr after infection, unintegrated DNA was isolated from the cells and analyzed for viral DNA by agarose gel electrophoresis, transfer to nitrocellulose paper, and hybridization with a radioactive RSV-specific probe (Southern, 1975; Shank et al., 1978). Figure 6 shows that the loss in infectivity after HMT treatment correlated with a decrease in the amount and size of unintegrated viral DNA present in viva.

DISCUSSION

Our results show that the RSV genome contains regions of secondary structure


that can react with several different psoralen derivatives. Presumably the different derivatives act at similar sites in the RNA, although the nucleotide specificity of the different derivatives has not been evalu- ated. The crosslinking of the subunits of virion RNA is consistent with the presumption that the RSV genome is a complex within the virion and that the complex does not arise by aggregation of subunits during isolation (Coffin, 1979). The ability of psoralen compounds to penetrate virions and crosslink the RNA complex in situ provides a method for a more detailed determination of the structure of virion RNA. Our initial examination of crosslinked RNA by electron microscopy suggests that the RNA subunits interact at numerous points along their lengths (unpublished observation).

In addition to crosslinking the subunits together, the psoralen derivatives were able to “mask” the presence of covalent breaks in viral RNA. Assuming that these breaks occur at random positions along the RNA chain, then regions of secondary structure capable of reacting with these

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derivatives must be distributed over much of the subunit RNA in vitro and within the, virions. This conclusion is consistent with physical studies of retrovirus RNA that have indicated that as much as 59% of the RNA chain may be involved in secondary structure (Cavalieri, 1974).

The reaction of psoralen with virions has a profound effect on infectivity. In the presence of long-wavelength uv light, HMT treatment reduced the focus-forming titer of RSV over 4 orders of magnitude in a 30- min exposure (Fig. 3). The reactivity of psoralens is thought to be specific for nucleic acids as opposed to nucleic acid-protein or protein-protein interactions (Fred- eriksen and Hearst, 1979). Our results showing that polymerase activity is essentially unaltered by HMT treatment sup- port this view. In addition to inactivating RSV, psoralen derivatives have been shown to inactivate several other RNA containing viruses (Hearst and Thiry, 1977; Han- son et al., 1978; Nakashima et aZ., 1979).

The photochemical reaction of HMT with RSV in the presence of long-wavelength uv light produces an effect on viral

- Time of Irradiation (min)

FIG. 6. Viral DNA synthesis in viva after infection with HMT-treated RSV. Virus-containing medium (Prague A RSV) was photoreacted with HMT for the indicated periods of time. An aliquot was removed at each time point and a portion of the aliquot was titered for virus infectivity as measured by the focus-forming titer. The remainder of each aliquot was used to infect cultures of chicken embryo fibroblasts. Unintegrated DNA was prepared from cells 8 hr after infection and viral DNA was analyzed by gel electrophoresis as described in Materials and Methods. (A) Titer of focus-forming units of virus as a function of exposure to HMT and uv light. The initial titer was 7.7 x lo5 focus-forming units/ml. (B) Analysis of viral DNA by agarose gel electrophoresis, transfer to nitrocellulose paper, and hybridization with a RSV-specific probe. The mark on the left side shows the position of unintegrated linear viral DNA (Shank et & 1978). Lane 1 is DNA from uninfected chicken cells; lane 2 is DNA from cells infected with untreated virus; and lanes 3-6 are DNA samples from cells infected with virus which had been photoreacted with HMT for 3, ‘7, 10, and 13 min, respectively.


replication that is similar to the effect produced by irradiation with short-wavelength uv light. RSV focus-forming activity is lost as a function of exposure to short-wavelength uv light (Rubin and Temin, 1959). The decrease in infectivity cannot be accounted for by inactivation of the viral polymerase (Lovinger et aZ., 1975; Owada et aZ., 1976), yet the amount of viral DNA present in cells infected with irradiated virus is reduced (Bister et aZ., 1977). It has been suggested that the decrease in infectivity is a result of uv damage to the RNA template (Bister et al., 1977), although there is evidence for uv-induced crosslinks between virion proteins and genome RNA (Owada et aZ., 1976).

step very close to the initiation of synthesis, whereas our results indicate that psoralen is affecting synthesis by terminating chain elongation. At present we do not understand the molecular basis for the apparent difference in the mechanism by which psoralen derivatives affect these two systems.

ACKNOWLEDGMENTS

Concomitant with the loss of infectivity of HMT-treated virions is a decrease in the size and amount of viral DNA synthesized in vitro and in viva (Figs. 4-6). We attribute the loss of infectivity to photochemical derivatization of the RNA template making it unsuitable for synthesis of full length DNA. Since viral DNA synthesis is initiated at a unique position on the RNA template (Bishop, 1978), the ap- pearance of new discrete species of DNA synthesized in vitro suggests that the addition of psoralen to the RNA template causes termination of DNA synthesis at specific sites. In another report we will describe the positions of specific psoralen- induced terminations and discuss the im- plication of these terminations to features of secondary structure in viral RNA (manuscript in preparation). Other fac- tors could also contribute to the loss of infectivity, for example, inefficient pene- tration or uncoating of virions containing crosslinked RNA.

The mechanism of psoralen-induced inhibition of synthesis appears to be fun- damentally different between RSV and reovirus. For RSV we have shown the presence of truncated DNA products when HMT-treated RNA is used as template. Nakashima et al. (1979) have reported that AMT-treated reovirus supported the synthesis of reduced levels of RNA but that the overall size of the RNA products re- mained unchanged. The results with reovirus suggest that psoralen is acting at a

We thank Harold E. Varmus, Richard Parker, and John Majors for helpful discussions and B. Cook for excellent stenographic assistance. This work was supported by USPHS Grants CA 12705 and CA 19287 and Training Grant lT32 CA 09043 and American Cancer Society Grant MV48G to J.M.B. and Harold E. Varmus; USPHS Grant GM 11180 to J.E.H.; and USPHS Postdoctoral Fellowship 6 F22CA02119-011 from the National Cancer Institute to L.M.H. A pre- liminary report of this work was presented at the 1978 Cold Spring Harbor RNA Tumor Virus Meeting.

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