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The 50 to 70S RNA of the Harvey sarcoma-Moloney leukemia virus (MLV) complex consists of 30 to 40S RNA subunits of two different size classes and contains sequences homologous to Moloney mouse leukemia virus and to information contained in a C-type rat virus, termed NRK virus. We have isolated by preparative gel electrophoresis the large (component 1) and the small (component 2) 30 to 40S RNA species from the Harvey sarcoma-MLV complex. Harvey RNA component 1 was completely complementary to DNA transcribed from MLV RNA and showed no homology to DNA transcribed from NRK virus when annealed under conditions of DNA excess. Harvey RNA component 2 was about 65% complementary to MLV DNA and about 33% complementary to NRK virus DNA. Approximately 60 to 80% of the MLV-specific sequences in RNA component 2 is either a distinct molecular species or is part of a hydrid molecular including NRK virus- and MLV-specific sequences. The rest of the MLV sequences in component 2 could be accounted for by degraded component 1 co-purifying with component 2. The possible role of these sequences in the ability of the virus to transform cells is discussed.

The poly(A) sequence of 30 to 40 S Rous sarcoma virus RNA, prepared by digestion of the RNA with RNase T 1 , showed a rather homogenous electrophoretic distribution in formamide-polyacrylamide gels. Its size was estimated to be about 200 AMP residues. The poly(A) appears to be located at or near the 3′ end of the 30 to 40 S RNA because: (i) it contained one adenosine per 180 AMP residues, and because (ii) incubation of 30 to 40 S RNA with bacterial RNase H in the presence of poly(dT) removed its poly(A) without significantly affecting its hydrodynamic or electrophoretic properties in denaturing solvents. The viral 60 to 70 S RNA complex was found to consist of 30 to 40 S subunits both with (65%) and without (approximately 30%) poly(A). The heteropolymeric sequences of these two species of 30 to 40 S subunits have the same RNase T 1 -resistant oligonucleotide composition. Some, perhaps all, RNase T 1 -resistant oligonucleotides of 30 to 40 S Rous sarcoma virus RNA appear to have a unique location relative to the poly(A) sequence, because the complexity of poly(A)-tagged fragments of 30 to 40 S RNA decreased with decreasing size of the fragment. Two RNase T 1 -resistant oligonucleotides which distinguish sarcoma virus Prague B RNA from that of a transformation-defective deletion mutant of the same virus appear to be associated with an 11 S poly(A)-tagged fragment of Prague B RNA. Thus RNA sequences concerned with cell transformation seem to be located within 5 to 10% of the 3′ terminus of Prague B RNA.

The RNA of a replication-defective (rd) mutant, isolated from stocks of nondefective (nd) Schmidt-Ruppin Rous sarcoma virus of subgroup A (SR-A) and termed SR-N8, was compared to the RNAs of SR-A, of a transformation-defective derivative of SR-A (td SR-A) and of rd Bryan Rous sarcoma virus, RSV (minus). The molecular mass of the 30-40S species of SR-N8 RNA was estimated to be 21% (congruent to 7.5 to 8 times 10-5 daltons) smaller than that of SR-A by (i) electrophoresis in polyacrylamide gels and (ii) analyses of RNA complexity based on RNase T1-resistant oligonucleotides. ST-N8 shares probably all (=14) of its large RNase T1-resistant oligonucleotides with the RNA of SR-A as judged from the chromatographic distribution and the RNase A-resistant fragments obtained from RNase T1-resistant oligonucleotides. However, SR-N8 RNA lacked six large oligonucleotides which were present in the RNAs of SR-A and td SR-A. Conversely, the RNAs of SR-A, and of SR-N8 contained two oligonucleotides not found in td SR-A. The RNA of SR-N8 was found to differ from that of RSV (minus) in its electrophoretic mobility and its fingerprint pattern. It is concluded that the RNA of SR-N8 was generated by a deletion of SR-A. The extent of this deletion is compatible with the notion that the genetic information for the large viral envelope glycoprotein (molecular mass = 70,000-85,000 daltons) has been lost from the RNA of SR-A to yield SR-N8 RNA. From a comparison of td and rd deletion mutants, it appears that loss of different functions corresponds to the absence of different oligonucleotides in their RNA.

The ribonucleoprotein (RNP) internal components of influenza virus were separated into distinct size classes by sedimentation in glycerol gradients and examined by electron microscopy by using positive staining with uranyl acetate. The large RNP have a peak in length distribution at 90 to 110 nm, the medium, at 60 to 90 nm, and the small, at 30 to 50 nm. These lengths can be correlated with the estimated molecular weights of the ribonucleic acids contained in the various RNP size classes. The RNP structure appears to consist of a strand which is folded back on itself and coiled in a regular double-helical arrangement.

Class a and class b 30 to 40 S RNA subunits obtained by heat dissociation from the 60 to 70 S RNA complex of avian tumor viruses were compared with several RNA standards by electrophoresis in formamide-polyacrylamide gels. Class a RNA was found to have a lower electrophoretic mobility and hence probably a higher molecular weight than class b RNA. The absolute molecular weight of class a and b RNA could not be determined with accuracy, because the relationship between logarithm of molecular weight and mobility of the RNA standards was not linear. The size of class a RNA fell into the range of 2.4 × 10 6 to 3.4 × 10 6 daltons and that of class b into the range of 2.2 × 10 6 to 2.9 × 10 6 daltons, depending on the standards used. The possible biological significance of this difference is discussed.

Earlier electrophoretic analyses have shown that the 60-70S RNA of avian sarcoma viruses contains a characteristic subunit, termed class a subunit, which has a lower electrophoretic mobility than class b subunit found in transformation-defective derivatives of sarcoma viruses and in avian leukosis viruses. We have compared the RNAs of three nondefective avian sarcoma viruses, B77 and Prague and Schmidt-Ruppin strains of Rous sarcoma virus, with those of their transformation-defective ( td ) derivatives, td B77, td PR-C, and td SR-A, respectively, to determine the chemical basis for the difference between class a and b subunits. It was found by “fingerprinting” that ( 1 ) all (about 20-25) large T1 RNase-resistant oligonucleotides present in class b subunits of transformation-defective viruses have homologous counterparts in the class a subunits of corresponding nondefective sarcoma viruses and that ( 2 ) class a subunits contain a few (one or two) additional oligonucleotides that are not present in class b . By contrast the oligonucleotide fingerprints of avian tumor viruses of different strains and subgroups were very different. Cross hybridization of classes a and b RNA of sarcoma virus B77 with DNA transcribed from a corresponding transformation-defective virus td B77 showed that the two RNAs share at least 60% and differ by about 10% of their sequences. It is suggested that the structural relationship of class a and b subunits of corresponding viruses may be expressed as a = b + x , and that all the oligonucleotides present only in RNAs of sarcoma viruses but not in transformation-defective viruses of the corresponding strains are part of sequence(s) x . The possibility that x represents genetic information directly or indirectly involved in transformation of fibroblasts is discussed.

ADENYLIC ACID (A)-rich sequences have been found in some mRNAs of animal cells1,2 and DNA viruses3 but not in the RNAs of cytocidal or oncogenic RNA viruses. These sequences have been proposed to be involved in the transport of mRNA to the cytoplasm from the nucleus or vaccinia virus core2,3, or in the association of mRNA with proteins necessary for translation1.

Ribonuclease-resistant ribonucleic acid (RNA) was isolated from uridine-labeled cultures of rabbit kidney, chicken embryo, and HeLa cells. This RNA, regardless of its source, was found to induce interference with virus growth in either rabbit kidney or chicken embryo cultures. Nuclease-treated cellular nucleic acids exhibited interference-inducing activity which eluted with a small fraction of RNA in the exclusion volume of a 6% agarose gel column. Besides resistance to ribonucleases, the interference inducer and RNA isolated from partially digested nucleic acids have in common two properties of double-stranded RNA: (i) similar sharp melting profiles were obtained for inducer and ribonuclease-resistant RNA, with T m dependent on NaCl concentration; (ii) ribonuclease-resistant inducer and RNA banded together in Cs 2 SO 4 density gradients at a density characteristic of known double-stranded RNA. After melting at low ionic strength, the labeled RNA shifted to a higher density and its capacity to inhibit virus replication was lost. Velocity sedimentation analysis of the cellular ribonuclease-resistant RNA indicated that the majority sedimented between 7 and 11 S , but only RNA sedimenting at ≧8 to 20 S had a high specific activity of interference induction. Without prior ribonuclease treatment, the ribonuclease-resistant RNA can be precipitated with 2 m LiCl and thus appears to exist in purified cellular nucleic acids as part of molecular complexes with both single- and double-stranded regions of RNA. The biosynthesis of cellular double-stranded RNA is inhibited by actinomycin D.

Two glycoprotein components containing glucosamine, galactose, and fucose were isolated from different strains of Rous sarcoma virus (RSV) and from Rauscher mouse leukemia virus (MLV). The molecular weight of the major glycoprotein of RSV was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) to be between 90,000 and 105,000 daltons, depending on the strain of RSV. The minor glycoprotein had an approximate molecular weight of 37,000. Both glycoproteins have higher molecular weights than the proteins of the group-specific antigen of avian tumor viruses. The glycoproteins represent between 10% and 20% of the radioactive protein of purified virus. Disruption of RSV with SDS yields glycoprotein components with sedimentation coefficients between 4 S and 2 S. The glycoprotein derived from Tween 20 disrupted virus was obtained in a 4-2 S form and in an 8 S form. The 8 S component consists predominantly of a multimeric aggregate of the two viral glycoprotein components, and some group-specific antigen. Because both the 8 S and the 4-2 S glycoprotein derived from Tween 20 disrupted RSV specifically inhibited virus neutralizing antibody and because the glycoproteins of different strains of RSV had different electrophoretic mobilities, it was concluded that the glycoproteins of avian tumor viruses are part of or identical with the viral type- or subgroup-specific antigen.

The proteins of influenza (WSN) and Sendai virus have been separated by polyacrylamide gel electrophoresis into five components. In both cases, three of these components were shown to be glycoproteins containing fucose, galactose, and glucosamine. Two protein components of each virus were probably free from these sugar residues, including the structural unit of the viral ribonucleoprotein (molecular weight of about 60,000 daltons).

The 60–70S RNAs of several transforming and nontransforming avian tumor viruses have different electrophoretic mobilities. The RNA of transforming viruses contains two electrophoretically separable subunit classes: a and b . The relative concentrations of these subunits vary with the virus strain. Avian leukosis viruses and nontransforming derivatives of a sarcoma virus lack subunits of class a . It is suggested that the presence of the class a subunit is related to the transforming ability for fibroblasts of the virus.

The glycoproteins of several Rous sarcoma virus (RSV) strains were found to have a slightly lower electrophoretic mobility in polyacrylamide gels than those of corresponding leukosis viruses of the same subgroup and strain. The glycoproteins of T5, a temperature-sensitive mutant of Schmidt-Ruppin RSV affecting the transforming ability of the virus, had a lower electrophoretic mobility in virus propagated at the permissive temperature than in virus propagated at the nonpermissive temperature. Further the glycopeptides of viruses released from transformed and nontransformed cell were compared. Viral glycopeptides were prepared by exhaustive pronase digestion of viral glycoproteins. It was found that the glycopeptides of all viruses released from transformed cells were larger (MW ≈ 5100) than those of viruses (MW ≈ 3900) released from normal cells. These differences were not strain- or subgroup-specific, as all viruses belonging to different subgroups and strains, which were released from transformed cells, had large glycopeptides. The same was true for glycopeptides, which were all small, of viruses released from phenotypically normal cells. The roles of cellular and viral genes in determining the size of viral glycoproteins and glycopeptides are discussed.

The DNA polymerase of the Prague strain of Rous sarcoma virus of subgroup C and of the Schmidt-Ruppin strain of subgroup A has been solubilized. DNA polymerase purified by sucrose gradient sedimentation and chromatography on DEAE-cellulose represented less than 2% of the soluble [ 14 C]protein of the virus. The enzyme was separated from 90% of the viral glycoprotein; it is probably different from the viral group-specific antigen. The sedimentation coefficient ( s 20, w ) of the soluble DNA polymerase was 8 S before, and 6 S after, incubation with pancreatic RNase. The molecular weight of the 8S DNA polymerase was estimated to be about 170,000, and that of the 6S DNA polymerase to be about 110,000. Purified DNA polymerase had a high activity with 60-70S viral RNA or salmon DNA as template, but it had a low activity with heat-dissociated 60-70S RNA, influenza virus RNA, or the RNA of tobacco mosaic virus as template. Neither the 8S nor the 6S DNA polymerase had endogenous template activity. The DNA-dependent and the RNA-dependent DNA polymerase activities of the Prague strain coincided in sucrose gradients, both in the 8S and the 6S form. It is concluded that the RNA-dependent and the DNA-dependent DNA polymerase activities of the avian tumor viruses are probably due to the same enzyme.

Rous sarcoma virus harvested from cells at intervals of 3 min has the same density, sedimentation coefficient, and DNA polymerase as virus harvested at hourly intervals. The RNA of the Prague strain-C consists of a minor class of 60-70S RNA, a major class 30-40S RNA, and a 4-12S class of RNA present at variable concentration. The RNA of the Schmidt-Ruppin strain-A contains more 60-70S than 30-40S RNA. Upon incubation of virus harvested at 3-min intervals at 40° in cell growth medium or Tris-saline, most of the 30-40S RNA is converted to 60-70S RNA. The electrophoretic mobility of the 30-40S RNA of the Rous virus harvested at 3-min intervals is lower than that of the 30-40S subunits of completely dissociated 60-70S RNA; after heating, their mobilities are identical. Heating also releases some small RNAs from 30-40S RNA of virus harvested at 3-min intervals, but five times more 4S RNA is released if the 30-40S RNA is allowed to convert to 60-70S in the virus. The template activity for Rous virus DNA polymerase of the 30-40S RNA of Rous virus harvested at 3-min intervals is about five times lower than that of 60-70S RNA. It is suggested that association of 30-40S RNAs with some RNAs of the 4-12S class may take place simultaneously with their conversion to 60-70S RNA.

Double-stranded RNA has been isolated from chick cells and chick cells infected with vaccinia virus. The double-stranded vaccinia virus RNA induces production of interferon.

Extraction of the nucleic acid from virions followed by sedimentation reveals a major RNA species with a sedimentation coefficient of 60–70S.

The template activities of the 60-70S RNA complex and of the 30-40S subunit RNA species of Rous sarcoma virus were tested in a cell-free protein-synthesizing system from mouse ascites Krebs II cells. Stimulation of protein synthesis over the endogenous background was about 2-fold with 30-40S viral RNA and about 1.3-fold with 60-70S viral RNA as template. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the predominant polypeptide synthesized in vitro in response to 30-40S RNA of Rous sarcoma virus had a molecular weight of 75,000-80,000. This polypeptide could be precipitated by antiserum against the group-specific antigens of the virus, although its molecular weight is higher than that of virion group-specific antigen proteins. Analysis of tryptic digests of the protein made in vitro indicates similarity to tryptic digests from authentic virion group-specific proteins. It is concluded that part of the RNA from Rous sarcoma virus is translated in vitro into a high-molecular-weight protein, perhaps a precursor of the virion group-specific proteins.

The structure and molecular weight of the 60-70S RNA complex and the 30-40S RNA species of Rous sarcoma virus were analyzed in an electron microscope after treatment of the RNAs with the bacteriophage T4 gene-32 protein to stretch out the RNA strands. Although all RNA preparations treated with gene-32 protein showed considerable heterogeneity in length, a significant fraction of the RNA retained its original sedimentation coefficient after treatment to allow the following conclusions to be made: The 30-40S RNA was confirmed to be a linear polynucleotide with a molecular weight of about 3 × 10 6 . The 60-70S RNA exhibited a network structure with a molecular weight predominantly of about 6 × 10 6 . Therefore, the subunit hypothesis for the 60-70S RNA is confirmed. A model for the structure and molecular weight of the 60-70S RNA postulates that the complex consists of two 30-40S RNA subunits held together at many points. This model elucidates the biological observation that the infectivity of RNA tumor viruses is proportional to the amount of 30-40S RNA in a virus preparation and not to the amount of 60-70S RNA.

The RNAs of several avian tumor virus recombinants that had inherited their focus-forming ability from a sarcoma virus and their host range marker from a leukosis virus were investigated. Electrophoretic analyses showed that the cloned sarcoma virus recombinants contained only size class a RNA, although they had acquired a marker that resided on class b RNA in the leukosis virus parent. Class a RNA of different recombinant clones, derived from the same pair of parental viruses and selected for the same biological markers, differed slightly in electrophoretic mobility from each other and from the parental sarcoma virus. They were also found to have different fingerprints of RNase T1-resistant oligonucleotides. The average complexity of the 60-70S RNA prepared from Prague Rous sarcoma virus of subgroup B was estimated to be 3.5 × 10 6 daltons from the size of 20 RNase T1-resistant oligonucleotides, which represented 3.9% of the RNA and that of a recombinant to be 3.3 × 10 6 daltons from 23 oligonucleotides, which represented 4.7% of the RNA. This result suggests that the genome of wild-type and of recombinant RNA tumor viruses is polyploid. The sum of these observations led us to propose that recombination among avian tumor viruses occurred by crossing-over between homologous pieces of nucleic acid.

Newcastle disease virus was found to contain three major proteins. The structure unit of the viral nucleocapsid appears to be monomeric and to consist of a single large protein of an approximate molecular weight of 62,000.

The interferon response may be induced by a variety of nonreplicating polyanionic macromolecules (Ho et al., 1967). Double-stranded RNA from a variety of sources is a potent inducer of interferon (Lampson et al., 1967; Tytell et al., 1967; Field et al., 1967a). The demonstration that double-stranded synthetic polyribonucleotides are also effective interferon inducers (Field et al., 1967b) stimulated the research presented in this paper.

Heating the 60 to 70 S ribonucleic acid (RNA) of Rous sarcoma virus (RSV) destroys both its subunit structure and its high template activity for RSV deoxyribonucleic acid (DNA) polymerase. In comparative analyses, it was found that the template activity of the RNA has a thermal transition of 70 C, whereas the 60 to 70 S structure dissociates into 30 to 40 S and several distinct small subunits with a T m of 55 C. Analysis by velocity sedimentation and isopycnic centrifugation of the primary DNA product obtained by incubation of 60 to 70 S RSV RNA with RSV DNA polymerase indicated that most, but perhaps not all, DNA was linked to small (S ) RSV RNA primer. Sixty percent of the high template activity of 60 to 70 S RSV RNA lost after heat dissociation could be recovered by incubation of the total RNA under annealing conditions. The template activity of purified 30 to 40 S subunits isolated from 60 to 70 S RSV RNA was not enhanced significantly by annealing. However, in the presence of small (S ) subunits also isolated from 60 to 70 S RNA, the template activity of 30 to 40 S RNA subunits was increased to the same level as that of reannealed total 60 to 70 S RNA. It was concluded that neither the 30 to 40 S subunits nor most of the 4 S subunits of 60 to 70 S RSV RNA contribute much as primers to the template activity of 60 to 70 S RSV RNA. The predominant primer molecule appears to be a minor component of the S subunit fraction of 60 to 70 S RSV RNA. Its electrophoretic mobility is similar to, and its dissociation temperature from 60 to 70 S RSV RNA is higher than that of the bulk of 60 to 70 S RSV RNA-associated 4 S RNA. The role of primers in DNA synthesis by RSV DNA polymerase is discussed.

Kirsten murine sarcoma-leukemia virus (Ki-MSV[MLV]) was found to contain less RNase H per unit of viral DNA polymerase than avian Rous sarcoma virus (RSV). Upon purification by chromatography on Sephadex G-200 and subsequent glycerol gradient sedimentation the avian DNA polymerase was obtained in association with a constant amount of RNase H. By contrast, equally purified DNA polymerase of Ki-MSV(MLV) and Moloney [Mo-MSV(MLV)] lacked detectable RNase H if assayed with two homopolymer and phage fd DNA-RNA hybrids as substrates. On the basis of picomoles of nucleotides turned over, the ratio of RNase H to purified avian DNA polymerase was 1:20 and that of RNase H to purified murine DNA polymerase ranged between

Several natural RNAs were compared with respect to their template activities for the DNA polymerase of Rous Sarcoma Virus during a 2-hr incubation period. 60-70S viral RNA was found to be a 5- to 10-fold better template than heat-dissociated Rous viral RNA, influenza virus RNA, tobacco mosaic virus RNA, or ribosomal RNA. Denatured salmon DNA is a little better, and poly(dAT) is 2-4 times better as a template for the enzyme than is 60-70S Rous viral RNA. The 60-70S RNAs of different strains of avian tumor viruses have very similar template activities for a given avian tumor virus DNA polymerase. Oligo(dT) or oligo(dC) were found to enhance the template activity of heat-dissociated Rous viral RNA 20- to 30-fold, and that of other natural RNAs tested one- to several-fold. DNA syntheses of 1-24% were obtained during a 2-hour incubation of the enzyme with the above RNA templates. The results suggest that the enzyme prefers partially doublestranded or hybrid regions of RNAs for optimal DNA synthesis, but certain regions of single-stranded RNA can also serve as templates. Poly(dAT) competes with viral RNA for purified DNA polymerase during DNA synthesis, as would be expected if RNA- and DNA-dependent DNA synthesis was performed by at least one common active site of the same enzyme.

The proteins of purified avian tumor viruses, including the Bryan Rous sarcoma and Rous-associated virus (RSV (RAV)), avian myeloblastosis virus (AMV), the “defective” ∗ NP-particle (H. L. Robinson, 1967) which is also known as RSV(0) (Vogt, 1967) and the nondefective Schmidt-Ruppin RSV(SR-RSV) were isolated by the phenol method. Each amino acid-labeled virus was found to contain two major and one minor radioactive proteins. These three proteins correspond to about 60–80% of the total radioactivity of amino-acid labeled RSV(RAV), RSV(0), and SR-RSV. Polyacrylamide gel electrophoresis under two different conditions showed that each virus contained the same three proteins in similar relative amounts. The two major proteins had a high complement-fixation titer with antibody against the group-specific antigen of the avian leukosis viruses, suggesting that the group-specific antigen of the avian leukosis viruses consists of at least two proteins. The “defective” RSV(0) isolated from NP-cultures Hanafusa et al., 1963 , Hanafusa et al., 1964 ; H. L. Robinson, 1967) contained all the proteins of the group-specific antigen detectable by the described methods. From this it is concluded that the “defectiveness” of the Bryan strain of RSV is not based on a lack of the group-specific antigen in the virus particle.

RNA tumour viruses are divided on the basis of their biological and serological properties into three major groups: avian leukosis sarcoma, mouse leukaemia sarcoma and mouse mammary tumour1. Common features in electron microscopic appearance2–6, chemical composition7 and the presence of a group-specific antigen common to all members of the respective groups7, suggest that all the viruses may have similar structure. Addition of inhibitors of DNA-dependent RNA synthesis to infected cultures blocks virus production for members of all three groups (refs. 8–10 and unpublished results of R. C. Cardiff and P. B. Blair), so it is also probable that they have some characteristics of replication in common. Single-stranded RNA has been isolated from several of these viruses by the SDS-phenol method11–15. In each case density gradient sedimentation of the RNA yielded a fast-sedimenting component with sedimentation coefficient (Sw) of 60S-75S which was identified as the intact viral UNA, plus a 4S component which was thought to be degraded viral or cellular RNA.

Influenza virus A was disrupted by deoxycholate, Triton X100 or ether extraction into ribonucleoprotein and envelope proteins. The ribonucleoprotein was resolved into a 70 s, a 60 s and a 50 s component by sucrose gradient sedimentation. Three components of viral ribonucleoprotein were also obtained by polyacrylamide gel electrophoresis. The buoyant density of the viral nucleoprotein was 1.265 g/ml. in a sucrose density-gradient. The ribonucleoprotein was relatively resistant to pronase. But the RNA of the ribonucleoproteins of influenza virus was degraded by RNase. Probably the same three ribonucleoprotein components were also detected in virus-infected cells about three hours after infection. Distinct components of the influenza virus nucleoprotein were found to contain distinct viral RNA's, suggesting that their heterogeneity is original and not an artifact of the isolation procedures used.

particle as multiple pieces in certain cases.5 6 Recently, in this laboratory, the intact RNA was isolated from the mixture of Rous sarcoma virus (RSV) and its helper virus RAV which belong to the avian leukosis complex of viruses.7 This result suggested that similar methods be used with a member of the myxovirus group. This paper describes the purification of NDV and the isolation of a 57S2o RNA, one molecule of which probably accounts for the total RNA per virus particle. While this study was in progress a brief report appeared on the isolation of a 45-50S RNA from NDV.8 Materials and Methods.-The plaque-forming L-Kansas 48 strain of NDV was used in all experiments. Ten-day-old eggs were inoculated in the allantoic cavity with 1-3 X 102 PFU of virus. After incubation at 40?C for 40-44 hr, the eggs were chilled and the chorioallantoic fluid was harvested. To remove cell debris the fluid was centrifuged at 4000 rpm for 10 min in a Servall GSA rotor. The supernatant was then kept overnight at 4?C. Virus purification: The virus was purified in three steps, all carried out in the cold (0-4?C). The following procedure was used to purify 800-1800 ml of allantoic fluid. First step: Centrifugation to a density interface: Twenty-eight ml of allantoic fluid was layered on 5 ml of a sucrose-RbCl solution and centrifuged in the Spinco 30 rotor for I hr at 30,000 rpm. The sucrose-RbCl solution contained 60% sucrose, 15% RbCl, 0.01 M Tris hydroxymethylaminomethane HCl (Tris - HCl), pH 7.3, and 0.001 M versene (EDTA); its density was 1.34gm per ml. After centrifugation a visible band of virus was seen at about density 1.22 gm per ml in the steep density gradient between the sucrose-RbCl solution and the allantoic fluid. A second band of nonviral material appeared 3-5 mm above the virus zone at about density 1.04 gm per ml. The tube was then punctured at the bottom, the virus band collected, and the upper band of foreign material discarded. The virus solution was diluted with an equal volume of buffer, 0.01 M Tris HCI, pH 7.3, 0.001 M EDTA and 1/4 vol of medium containing 4% calf serum.9 Second step: Ammonium sulfate precipitation: An equal volume of saturated ammonium sulfate, previously neutralized with Tris HCI buffer, was added dropwise to the virus solution. The precipitate was collected by centrifugation, and the supernatant containing about 1% of the starting infectivity was discarded. Third step: Sucrose-D2O gradient: The precipitate was redissolved in buffer containing 0.01 M Tris HCl, pH 7.3, 0.1 M NaCl, and 0.001 M EDTA to make a final volume between 4 and 9 ml depending on the amount of virus present to ensure that the solution density was less than 1.07 gm per ml. Up to 4.5 ml of the virus solution was then layered on top of an 18-ml linear gradient of sucrose in heavy water and then overlaid with mineral oil to fill the tube and centrifuged in a Spinco SW25 rotor at 25,000 rpm for 6 hr. The gradient was made, using 65% sucrose in D20 (density 1.32 gm per ml) and an equal volume of that solution diluted 4-fold with H20. Both solutions and the final gradient contained 0.01 M Tris-HC1, pH 7.3, 0.05 M NaCl, and 0.001 M EDTA. The virus band could be seen at about the middle of the tube. A second band of nonviral ma

Influenza virus PR8 has been purified from the allantoic fluid of infected chick embryos and from tissue culture medium. Preliminary characterization indicates that the virus contains at least two major proteins and several RNA components with sedimentation coefficients ( S 20, w ) ranging from about 9 to 18 s. Synthesis of virus-specific RNA in influenza virus-infected chick embryo cells has been demonstrated under conditions where cell RNA synthesis is suppressed by actinomycin D. In addition to the single-stranded viral RNA components, a smaller fraction of virus-specific double-stranded RNA is labeled with [ 3 H]-uridine in the presence of actinomycin D. This RNA has been purified after incubation of the RNA from infected cells with RNase. It has an approximate sedimentation coefficient of 8 to 10 s, shows a sharp thermal transition and has a lower buoyant density in cesium sulfate than single-stranded influenza virus RNA. Preliminary evidence indicates that the double-stranded RNA may be an intermediate in viral RNA synthesis.

The 5′ terminus of the Rous Sarcoma Viral 30-40S RNA was characterized as follows: Unlabeled RNA was treated with polynucleotide kinase and (γ-32P) ATP. Degradation of the 5′-(32P) RNA with alkali yielded labeled pAp while degradation with venom phosphodiesterase yielded labeled 5′-AMP. Dephosphorylation with alkaline phosphatase was unnecessary for the RNA to accept32P indicating the presence of 5′-OH ends. This establishes that the base at the 5′ end of Rous Sarcoma Viral 30-40S RNA is adenine.

Summary Indirect evidence suggests that the replication of RNA tumor viruses differs from that of cytocidal RNA viruses in two distinctive properties: a requirement for early DNA synthesis and one for permanent DNA dependent RNA synthesis. These requirements led to the provirus hypothesis of Temin which states that there is a DNA intermediate in RNA tumor virus replication. The provirus hypothesis has been strengthened by the discovery of virus-associated RNA dependent DNA polymerase. Yet, neither provirus hypothesis nor the virus-associated RNA dependent DNA polymerase explain the peculiar requirement of RNA tumor virus replication for cellular DNA synthesis. Numerous recent observations suggest that some RNA tumor virus genes, in some cases even all viral genes, preexist in normal cells. If these genes exist in phenotypically normal cells as unexpressed DNA, it is conceivable that “cellular” DNA synthesis is required in RNA tumor virus replication to derepress these genes. In this case the genetic role of the virus or of the viral RNA in RNA tumor virus infection becomes a philosophical question.

Preinfection of chick embryo fibroblasts with an avian tumor virus does not eliminate the requirement of early DNA synthesis for other avian tumor viruses superinfecting the same cells several days after the primary inoculum. The function served by the early DNA in avian tumor virus infection either fails to persist into late infection or is genetically specific for each viral type.

An improved procedure for the purification of mouse encephalitis virus is described. The protein isolated from such purified preparations exhibited several electrophoretic components even after attempts to remove possible mutant or companion viruses by careful clonal purification of the parent stocks. All of these protein components, therefore, evidently derive from a single genotypic class of virus particles. The two major protein components were isolated by preparative electrophoresis on Polyacrylamide gel and found to differ in both amino acid content and in tryptic mapping pattern. The slower of the two major components, furthermore, appeared on the basis of its electrophoretic profile and tryptic map to be a partially resolved mixture. It is concluded that mouse encephalitis virus contains at least two and very probably three different major polypeptide chains.