Synthesis of Saint Louis Encephalitis Virus Ribonucleic Acid in BHK-21/13 Cells

Infection of baby hamster kidney cells (BHK-21/13) with Saint Louis encephalitis (SLE) virus depressed the rate of protein and ribonucleic acid (RNA) synthesis until viral RNA synthesis began 6 hr postinfection (PI). Virus-directed RNA synthesis was subsequently inhibited until 12 hr PI when virion maturation began. The rate of protein synthesis reached a peak 6 hr PI and was subsequently depressed until just before the onset of virion maturation. Density gradient analysis of phenol-extracted RNA from actinomycin-treated infected cells indicated that, at 6 to 8 hr and again at 12 to 20 hr PI, three species of viral-specific RNA were synthesized. The most rapid sedimenting form (43S) was ribonuclease-sensitive and had a base composition similar to the RNA isolated from mature virions. The 20S RNA species was ribonuclease-resistant and had a sedimentation coefficient and base composition similar to the replicative form associated with other arbovirus infections. The 26S RNA was ribonuclease-resistant (0.2 mug/ml, 0.1 m NaCl, 25 C, 30 min) and had a nucleotide base composition closer to the 20S form than to the values for 43S RNA. Five-minute pulse labeling of infected cultures during the period viral RNA synthesis was maximal resulted in labeling of only the 20S to 22S RNA fractions. With pulse-labeling periods of 10 min, both the 20S and 26S RNA species were radioactive. Periods of radioactive labeling of as long as 15 min were required before the 43S form was radioactively labeled. These results suggest that the 20S and 26S RNA may be intermediate forms in the synthesis of 43S viral RNA.     

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus.

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus were studied. Electron micrographs of the infected cells showed that most of mutant virions bud from the plasma or vacuolar membrane as a multiploid particle containing a various number of nucleocapsids enclosed with a defined common envelope. The mutant virions contained three polypeptides which migrated to the position identical with those of wild type on discontinuous acrylamide gels. Cells infected with the mutant virus synthesized the same intracellular viral RNA species as was made after infection of wild type. Cytoplasmic nucleocapsids of the mutant sedimented at 140S and contained 42S virion RNA as those of wild type; they were indistinguishable from those of wild type in an electron microscope examination. On the other hand, mutant nucleocapsids isolated from extracellular virions sedimented as heterogeneous particles larger thant 140S and were shown to be pleomorphic and aggregate in electron micrographs. The budding process of this mutant seemed to be modified, so that it might form the multiploid with the alteration of its nucleocapsids.     

Electrophoretic Characterization of Foot-and-Mouth Disease Virus-Specific Ribonucleic Acid

Foot-and-mouth disease virus (FMDV)-specific ribonucleic acid (RNA) was analyzed by electrophoresis on 0.5% agarose gels. Four classes of RNA were resolved as a function of mobility in agarose: two classes of slowly migrating multistranded RNA, the infectious viral RNA with intermediate mobility, and a minor fast-moving class of lower-molecular-weight single-stranded RNA. The major RNA species were infectious viral RNA and the slowest migrating class of multistranded RNA. The latter RNA was polydisperse when analyzed by sucrose gradient centrifugation, it was partially ribonuclease resistant, and it was the predominant RNA species labeled during the initial period of (3)H-uridine triphosphate incorporation in the cell-free system. Heat treatment studies indicated that part of the slowest-moving RNA was degraded at 60 C and almost complete degradation was detected at 100 C. It was concluded that this RNA is the replicative intermediate in viral RNA synthesis. The second class of multistranded RNA contained both a ribonuclease-resistant RNA and a second RNA peak which was detected only after heat treatment at temperatures above 75 C. Fractions of FMDV-specific RNA isolated by sucrose gradient centrifugation were analyzed by agarose-gel electrophoresis. Infectious viral RNA was detected only in the 37S zone and was the major species of RNA in this part of the gradient. The ribonuclease-resistant RNA (the 20S zone) contained about equal amounts of multistranded RNA (both classes) and the low-molecular-weight single-stranded RNA. All sucrose gradient fractions between 20 and 40S were found to contain the replicative intermediate, although the major portion was detected in the 20 to 25S region.     

Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.     

Replication of Western Equine Encephalomyelitis Virus II. Cytoplasmic Structure Involved in the Synthesis and Development of the Virions

Analysis of the cytoplasmic fraction of chick embryo cells during the exponential phase of Western equine encephalomyelitis (WEE) virus growth showed that the viral ribonucleic acid (RNA) labeled by a short pulse with ³H-uridine was associated with a structure which sedimented in sucrose density gradients with a coefficient of 65S. The RNA extracted from this structure sedimented in sucrose density gradients at 26S. After a longer period of exposure to ³H-uridine, the radio-active viral RNA was associated with a structure which sedimented in sucrose density gradients as would materials with coefficients of about 140S. The 140S structure contained viral RNA and viral protein. It was shown that the 140S structures are not virus-induced polysomes. The 140S structure contained predominantly the 40S type of viral RNA and some 26S type. Electrophoretic analysis of the disrupted virion revealed that at least two proteins (types I and II) were present in the purified virion. Only type II protein was present in the 140S structure. Unlike the virion, the 140S structure did not contain any lipid which could be detected by the incorporation of ¹⁴C-choline. These data suggest that the 140S structure represents the internal nucleoprotein part of the virion. The rate of appearance of labeled virus lags behind that of the formation of the 140S structure in infected cells. Pulse-chase experiments with ³H-leucine suggest that the 140S structure may represent a precursor to the virus particle. The results are discussed in terms of the maturation of WEE virus in the infected cells.     

Replication of Sendai Virus: II. Steps in Virus Assembly

Chick embryo fibroblast cultures infected with Sendai virus were incubated with (3)H-uridine in the presence of actinomycin D beginning at 18 hr after infection. The 35 and 18S virus-specific ribonucleic acid (RNA) components were found in a ribonuclease-sensitive form in the cell and appeared to be associated with polyribosomes. Newly synthesized 57S viral RNA was rapidly coated with protein to form intracellular viral nucleocapsid, and no 57S RNA was found "free" (ribonucleasesensitive) in the 2,000 x g supernatant fraction of disrupted cells. The nucleocapsid from detergent-disrupted Sendai virus and that from disrupted cells were indistinguishable in ultrastructure and buoyant density, and neither was found to be infectious or have hemagglutinating activity. Kinetic studies of nucleocapsid and virus formation indicated a relative block in conversion of viral nucleocapsid to complete enveloped virus in these cells, resulting in accumulation of large amounts of nucleocapsid in the cell cytoplasm.     

Basis for Variable Response of Arboviruses to Guanidine Treatment

The effect of guanidine on the replication of the group A arboviruses, Sindbis virus, and Semliki Forest virus (SFV) was studied. Guanidine rapidly, but reversibly, inhibited SFV ribonucleic acid (RNA) synthesis. The synthesis of all species of viral RNA was inhibited, but that of ribonuclease-resistant forms was least affected. This inhibition occurred when the drug was added at any point during the log phase of virus growth. The growth of SFV was also markedly inhibited, but Sindbis virus growth was unimpaired. Infection of guanidine-treated cells with the viruses together resulted in a significant inhibition of the yields of both. It appears that, in the case of Sindbis virus, viral RNA is ordinarily produced in such excess that inhibition of its synthesis does not reduce virus yields. In the case of SFV, guanidine also markedly distorts the pattern of RNA synthesis by greatly decreasing the production of the 26S interjacent RNA form. This may account for the observed inhibition of SFV growth in the presence of guanidine.     

Chromatography of Arbovirus Ribonucleic Acid Forms on Columns of Benzoylated-Diethylaminoethyl Cellulose

Benzoylated-diethylaminoethyl cellulose (BD-cellulose) column chromatography was found to be useful in resolving most of the ribonucleic acid (RNA) forms from the replicative cycle of group A arbovirus Semliki Forect virus (SFV). The elution patterns were independent of molecular weight and appeared to be related to the degree of secondary structure in the molecule. Fractions of RNA were taken from a sucrose density gradient of cytoplasmic extracts of SFV-infected chick cells pretreated with actinomycin D. In a linear salt gradient, 16S material cochromatographed with the rapidly eluted ribonuclease resistant core of the double-stranded SFV-RNA and with the homopolymer duplex polyinosinic acid: polycytidylic acid. This fraction, therefore, probably contains an SFV-RNA form similar to the completely double stranded replicative form (RF) of several RNA viruses and bacteriophages. Faster moving (>20S) sucrose gradient fractions eluted more slowly, suggesting a decreasing proportion of secondary structure with increasing sedimentation value. The fractions, therefore, seemed to contain replicative intermediate (RI) structures. The two single stranded forms of SFV-RNA (42S and 26S) could only be eluted from BD-cellulose in the presence of urea or dimethyl sulfoxide, suggesting the presence of minimal secondary structure. Under these conditions, the single-stranded viral RNA forms could not be resolved. Molecular sieve chromatography of the single-stranded RNA forms, performed by passage through an agarose column, also failed to resolve these forms. The viral RNA forms containing a high degree of secondary structure, probably the RF and the RI, could, therefore, be rapidly separated from each other and from the single-stranded forms.     

Replication of Sindbis Virus V. Polyribosomes and mRNA in Infected Cells

Cells infected with wild-type Sindbis virus contain at least two forms of mRNA, 26S and 49S RNA. Sindbis 26S RNA (molecular weight 1.6 x 10(6)) constitutes 90% by weight of the mRNA in infected cells, and is thought to specify the structural proteins of the virus. Sindbis 49S RNA, the viral genome (molecular weight 4.3 x 10(6)), constitutes approximately 10% of the mRNA in infected cells and is thought to supply the remaining viral functions. In cells infected with ts2, a temperature-sensitive mutant of Sindbis virus, the messenger forms also include a third species of RNA with a sedimentation coefficient of 33S and an apparent molecular weight of 2.3 x 10(6). Hybridization-competition experiments showed that 90% of the base sequences in 33S RNA from these cells are also present in 26S RNA. Sindbis 33S RNA was also isolated from cells infected with wild-type virus. After reaction with formaldehyde, this species of 33S RNA appeared to be completely converted to 26S RNA. These results indicate that 33S RNA isolated from cells infected with either wild-type Sindbis or ts2 is not a unique and separate form of Sindbis RNA.     

Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid

Actinomycin D-treated chick fibroblasts were infected with purified (32)P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 mug/ml) or cycloheximide (200 mug/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of (32)P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA.     

Viral RNAs Associated with Ribosomes in Sindbis Virus-Infected HeLa Cells

Virus specific RNA ribosome complexes were isolated by sucrose density gradient centrifugation of cytoplasmic extracts from HeLa cells infected at 42 C with an RNA(+) mutant (ts2) of Sindbis virus. Viral RNA-ribosome complexes were accumulated by infected cells treated with sodium fluoride and cycloheximide. The RNA-ribosome complexes were characterized by (i) their sensitivity to the action of ribonuclease or ethylenediaminetetraacetic acid, (ii) their density in cesium chloride gradients, and (iii) presence of host ribosomes and viral RNAs. The viral RNAs were isolated and characterized. The results showed that two species of single-stranded RNAs (a 28s and 18 to 15s species) were associated with the complexes. Base composition analysis of the viral RNAs indicated that both species had a higher adenine content than the 42s or 26s forms of viral RNAs. The RNAs associated with the ribosome complexes were virus specific since they annealed with denatured double-stranded RNAs from the infected cells. Little or no 42S RNA was associated with the RNA-ribosome complexes. The results suggest that the 28s and 18 to 15s forms of RNAs may represent viral messenger RNAs.     

Replication of vesicular stomatitis virus defective interfering particle RNA in vitro: transition from synthesis of defective interfering leader RNA to synthesis of full-length defective interfering RNA.

The replication of the RNA of vesicular stomatitis virus (VSV) defective interfering (DI) particles was established in a defined cell-free system. The transition from synthesis of only the DI-leader RNA to replication of the full-length DI RNA was effected in the system by newly synthesized VSV proteins and occurred in the absence of VSV helper virus. Both positive- and negative-polarity full-length DI RNA were synthesized. Furthermore, the products of RNA replication associated with newly synthesized viral proteins to form complexes that were indistinguishable from authentic DI particle nucleocapsids on the basis of buoyant density and resistance to ribonuclease digestion. The DI-leader RNA did not form ribonuclease-resistant structures. We conclude that this in vitro system successfully executes many of the reactions of VSV DI particle replication and assembly.     

Properties of a Ribonucleoprotein Particle Isolated from Nonidet P-40-Treated Rous Sarcoma Virus

A ribonucleoprotein particle containing about 20% ribonucleic acid (RNA), and containing little if any phospholipid or glucosamine, was recovered in high yield after treatment of Schmidt-Ruppin strain of Rous sarcoma virus and B77 virus with the nonionic detergent Nonidet P-40. This structure, which probably derives from the internal ribonucleoprotein filament described in electron microscopy studies, contained 80 to 90% of the viral 60 to 70S RNA and only about 10% of the protein present in intact virions. It sedimented in glycerol density gradients at approximately 130S and had a buoyant density in sucrose of about 1.34 g/ml. Studies with (32)P-labeled virus indicated that the ribonucleoprotein particle contained approximately 30 4S RNA molecules per 10(7) daltons of high-molecular-weight viral RNA. Intact virions contained about 70 4S RNA molecules per 10(7) daltons of high-molecular-weight RNA. Electrophoretic studies in dodecyl sulfate-containing polyacrylamide gels showed that the ribonucleoprotein particle contained only 5 of the 11 polypeptides found in the virion; of these the major component was a polypeptide weighing 14,000 daltons.     

Effect of Actinomycin D on Virus-induced Ribonucleic Acid Polymerase Formation in Foot-and-Mouth Disease Virus-Infected Baby Hamster Kidney Cells

Actinomycin D, at a concentration that inhibits cellular ribonucleic acid (RNA) synthesis, inhibited the production of foot-and-mouth disease virus-induced RNA polymerase in baby hamster kidney cells. Inhibition was proportional to exposure time and reached 85% when actinomycin D was added 90 min before infection. Polymerase production was inhibited to the same extent in growth and minimal media, and the kinetics of its appearance were slightly different than in untreated cells. Enzyme preparations from actinomycin-treated cells having one-third to one-tenth the activity of untreated samples gave products with RNA profiles similar to those of controls. The 37S viral peak, 20S ribonuclease-resistant peak, and 26 to 28S peaks were present in all cases. Actinomycin D did not consistently inhibit virus production in either medium. Insulin did not prevent the actinomycin induced inhibition of polymerase and virus production from occurring.     

Initiation sites for translation of sindbis virus 42S and 26S messenger RNAs.

Sindbis virus 26S RNA is the principal species of virus-specific RNA found in the infected cell; it is derived from a one third segment of virion 42S RNA. When translated in cell-free extracts from mouse ascites cells or rabbit reticulocytes, 26S RNA directed the synthesis primarily of the 33,000 dalton virus capsid protein, and the protein products were in the form of free peptides rather than peptidyl-tRNA. In contrast, the polypeptides synthesized in either extract in response to Sindbis virus 42S RNA were heterogeneous, ranging in molecular weight from 33,000 to 190,000, and were largely in the form of peptidyl-tRNA. The number of independent initiation sites on the 26S and 42S RNAs was determined by analyzing a tryptic digest of reaction products labeled with yeast N-formyl-35S-methionyl-tRNAFmet. The 26S RNA appeared to contain a single initiation site, and this site could also be found in varying amounts in different preparations of 42S RNA. However, a second initiation site, distinct from that of 26S RNA, was the major site in 42S virion RNA. These results suggest that 42S virion RNA contains two potential sites for initiation of protein synthesis. Only one of these may be active, however, and it is postulated that the second site functions primarily, if not exclusively, in the subgenomic 26S RNA species. In this regard, Sindbis virus 42S RNA may represent a novel form of a eucaryotic messenger RNA.     

Two Classes of Cytoplasmic Viral RNA Synthesized Early in Productive Infection with Adenovirus 2

The RNA sequences and RNA size classes transcribed early in productive infection with adenovirus 2 were analyzed by RNA-DNA hybridization. Two independent procedures demonstrated that early cytoplasmic viral RNA is composed of two sequence classes, class I which is absent or present in greatly reduced quantities at 18 h, and class II which persists throughout the infection. When the sequences in early viral RNA were analyzed by hybridization-inhibition studies, the hybridization of early [(3)H]RNA was inhibited only 50% by RNA from cultures harvested late (18 h) in infection. Liquid hybridizations with radioactive viral DNA confirmed that early RNA includes two classes. Duplex formation of RNA with (32)P-labeled viral DNA was assayed by hydroxylapatite chromatography and resistance to S(1) nuclease digestion. Both methods showed that the cytoplasmic RNA present early in infection annealed 12 to 15% of the viral DNA; late cytoplasmic RNA hybridized 21 to 25% of the DNA. Mixtures of early plus late cytoplasmic RNAs hybridized 30 to 34% of the viral DNA, demonstrating the reduced concentration of early class I RNA in the late RNA preparations. Experiments were performed to correlate class I and class II early RNA with size-fractionated cytoplasmic RNA synthesized early in infection. Fractionation of RNA by gel electrophoresis or sucrose gradient centrifugation confirmed three major size classes, 12 to 15S, 19 to 20S, and 26S. Total cytoplasmic RNA and RNA selected on the basis of poly(A) content contained the same size classes of viral RNA. In standard electrophoresis conditions, the 19 to 20S viral RNA could be resolved into two size classes, and the distribution of 12 to 15S RNA also indicated the presence of more than one size component. Hybridization-inhibition studies under nonsaturating conditions were performed with 26S, 19 to 20S, and 12 to 15S viral RNAs fractionated by gel electrophoresis. Late RNA inhibited the hybridization of 26S RNA only 20%, 19 to 20S RNA was inhibited 45%, and 12 to 15S RNA was inhibited 50%. When 18 to 19S and 12 to 15S viral RNAs purified by sucrose gradient centrifugation were similarly analyzed, late RNA inhibited hybridization of 18 to 19S RNA 50%, and the annealing of 12 to 15S RNA was inhibited 70%.     

In Vitro Replication of Cowpea Mosaic Virus RNA I. Isolation and Properties of the Membrane-Bound Replicase

A fraction which contained the membrane-bound cowpea mosaic virus RNA replicase was isolated from cowpea mosaic virus-infected cowpea leaves. The replicase activity appeared on day 1 after inoculation, then increased to reach a maximal on day 4. The increase in enzyme activity preceded the most-rapid virus multiplication. The membrane-bound replicase activity was almost completely insensitive to actinomycin D and DNase. The corresponding fraction from healthy leaves had no RNA-dependent RNA polymerase activity. The viral RNA synthesis in vitro proceeded linearly for 20 min and required all four ribonucleoside triphosphates and Mg(2+) ions. Mn(2+) was a poor substitute for Mg(2+). The reaction was optimal at pH 8.2. During the whole period of RNA synthesis the in vitro synthesized RNA was at least 70% resistant against RNase in 2 x SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digestable by RNase in 0.1 x SSC. Analysis of the products by sucrose gradient centrifugation followed by treatment of separate fractions with RNase demonstrated that both single-and double-stranded RNA were present. Double-stranded RNA sedimented at about 20S, with a shoulder at 16S to 17S. A minor part of the double-stranded RNA sedimented below 10S. Single-stranded RNA sedimented with the same rate as the two viral RNAs, 26S and 34S.     

Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.

The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.     

Characterization of intracellular viral RNA in interferon-treated cells chronically infected with murine leukemia virus.

We have recently found that Moloney murine leukemia virus assembles within cytoplasmic vacuoles of chronically infected NIH/3T3 cells rather than at their surface (submitted for publication). In the present study we found that if these cells were treated with interferon (IF) for 24 to 48 h the intracellular virus particles accumulated at a two- to threefold-higher level than that observed in untreated cells. Nevertheless, despite this accumulation, no difference between IF-treated and untreated cells was observed in the amount of the total cytoplasmic viral RNA or in its 35S or 21S species. When cellular virions were sedimented from the cytoplasmic fraction, a markedly higher amount of viral RNA was detected in the viral pellet of IF-treated cells than was detected in untreated cells, whereas the amount of viral RNA left in the virus-free cytoplasm of IF-treated cells was much lower than that in the untreated cells. Furthermore, the amount of the cytoplasmic polyriboadenylic acid-containing viral RNA was also remarkably higher in the IF-treated cells. Viral polyribosomes appeared to be fully functional in IF-treated cells, since no effect of IF on viral protein synthesis could be detected. Analysis of the nuclear viral RNA showed no difference between IF-treated and untreated cells after 24 h of IF treatment. Both contained a comparable amount of 35S viral RNA. However, at 48 h a significant accumulation of viral RNA was observed in the nucleus of the IF-treated cells as compared with the untreated cells, although in both cases only 35S species were evident. This accumulation appeared to activate a degradation process which destroyed nuclear viral RNA, since a dramatic shift toward smaller-sized molecules of viral RNA and a remarkable reduction in its amount were observed after 72 h of IF treatment.     

Characterization of Intracellular Ribonucleic Acid Specific for the Murine Sarcoma-Leukemia Virus Complex

Virus particles were continuously produced by a cell line (78A1) of rat embryo fibroblasts that had been transformed by the murine sarcoma-leukemia virus complex. Since most of the mature virions were found in the extracellular fluid and were not cell-associated, a measurable quantity of viral ribonucleic acid (RNA) could not be extracted from these cells. Cycloheximide, a protein inhibitor, was successfully used to accumulate viral RNA within the cells. This ribonuclease-sensitive RNA, with a sedimentation coefficient of 71S, had the same base composition as the high molecular weight RNA (S(20,w) = 71) isolated from purified virions released by the transformed cells.