Isolation of a Transformation-Defective Deletion Mutant of Moloney Murine Sarcoma Virus

A transformation-defective (td) deletion mutant of Moloney murine sarcoma virus (td Mo-MSV) and a transforming component termed Mo-MSV 3 were cloned from a stock of clone 3 Mo-MSV. To define the defect of the transforming function, the RNA of td Mo-MSV was compared with those of Mo-MSV 3 and of another transforming variant termed Mo-MSV 124 and with helper Moloney murine leukemia virus (Mo-MuLV). The RNA monomers of td Mo-MSV and Mo-MSV 3 comigrated on polyacrylamide gels and were estimated to be 4.8 kilobases (kb) in length. In agreement with previous analyses, the RNA of Mo-MSV 124 measured 5.5 kb and that of Mo-MuLV measured 8.5 kb. The interrelationships among the viral RNAs were studied by fingerprinting and mapping of RNase T₁-resistant oligonucleotides (T₁-oligonucleotides) and by identification of T₁-oligonucleotides present in hybrids formed by a given viral RNA with cDNA's made from another virus. The nontransforming td Mo-MSV RNA lacked most of the Mo-MSV-specific sequence, i.e., the four 3′-proximal T₁-oligonucleotides of the six T₁-oligonucleotides that are shared by the Mo-MSV-specific sequences of Mo-MSV 3 and Mo-MSV 124. The remaining two Mo-MSV-specific oligonucleotides identified td Mo-MSV as a deletion mutant of MSV rather than a deletion mutant of Mo-MuLV. td Mo-MSV and Mo-MSV 124 exhibited similar deletions of gag, pol, and env sequences which were less extensive than those of Mo-MSV 3. Hence, td Mo-MSV is not simply a deletion mutant of Mo-MSV 3. In addition to their MSV-specific sequences, all three MSV variants, including td Mo-MSV, shared the terminal sequences probably encoding the proviral long terminal repeat, which differed from their counterpart in Mo-MuLV. This may indirectly contribute to the oncogenic potential of MSV. A comparison of td Mo-MSV sequences with either Mo-MSV 124 or Mo-MSV 3 indicated directly, in a fashion similar to the deletion analyses which defined the src gene of avian sarcoma viruses, that Mo-MuLV-unrelated sequences of Mo-MSV are necessary for transformation. A definition of transformation-specific sequences of Mo-MSV by deletion analysis confirmed and extended previous analyses which have identified Mo-MuLV-unrelated sequences in Mo-MSV RNA and other studies which have described transformation of mouse 3T3 fibroblasts upon transfection with DNAs containing the Mo-MSV-specific sequence.     

Translational Products of Moloney Murine Sarcoma Virus RNA: Identification of Proteins Encoded by the Murine Sarcoma Virus src Gene

In vitro translation of virion RNA of Moloney murine sarcoma virus (MSV) strain 124 yielded major products having molecular weights of 63,000 (63K), 43K, 40K, 31K, and 24K daltons. A molecularly cloned subgenomic fragment of Moloney MSV comprised of the cellular insertion (src) region was utilized in hybridization arrest translation as a means of identifying products of the MSV src gene. MSV src DNA specifically inhibited synthesis of the 43K, 40K, 31K, and 24K proteins, implying that each of these proteins was coded within the MSV src gene. The MSV src-specific nature of this family of proteins was further confirmed by partial purification of MSV src-containing RNAs from MSV non-producer cells. In vitro translation of enriched cellular RNAs yielded products with molecular weights identical to those of the 43K family of proteins synthesized from virion RNA. Nucleotide sequence analysis of the MSV transforming region has revealed a long open reading frame which includes five methionine codons (Reddy et al., Proc. Natl. Acad. Sci. U.S.A. 77:5234-5238, 1980). The molecular weights of the four largest proteins that could be synthesized within this open reading frame corresponded closely to the molecular weights of the 43K family of proteins. Partial cyanogen bromide cleavage of each of the three largest proteins resulted in an uncleaved fragment having a molecular weight equal to that of the smallest (24K) protein. These findings provide direct biochemical evidence that the 43K, 40K, 31K, and 24K proteins are related in their carboxy-terminal regions, as well as information concerning the MSV src gene coding sequences from which each protein originates:     

The 30S Moloney sarcoma virus RNA contains leukemia virus nucleotide sequences.

The 50S-70S RNA of a Moloney sarcoma-leukemia virus [Mo-MSV(MLV)] complex produced by a particular mouse cell line was shown by gel electrophoresis to contain a major (97%) 30S sarcoma-specific subunit species and a minor (3%) 38S leukemia virus-specific subunit. On the basis of its sedimentation coefficient and known complexity, the 30S Mo-MSV RNA was estimated to be a unique RNA molecule of about 6000 nucleotides. Hybridization experiments using viral RNA and DNA complementary to viral RNA (cDNA) made by viral DNA polymerase indicated that the 30S Mo-MSV RNA shared 70% of its sequences with Mo-MLV, 30% with another MLV derived from Mo-MLV, and 30% with Kirsten sarcoma-xenotropic leukemia virus. The 30S Mo-MSV RNA sequences shared with these viruses were not additive. The Tm of a Mo-MSV RNA-MLV cDNA hybrid was 83 degrees C, indicating that large contiguous nucleotide sequences were shared between the two nucleic acids. Mo-MSV RNA and Mo-MLV RNA shared possibly seven of 20-30 RNAase T1-resistant oligonucleotides, while Mo-MSV RNA contained three, and Mo-MLV RNA contained at least five specific oligonucleotides. We conclude that the 30S Mo-MSV RNA molecule consists of approximately 70% (about 4200 nucleotides) Mo-MLV-specific sequences and of 30% (1800 nucleotides) Mo-MSV-specific sequences covalently linked. Our results favor the hypothesis that 30S Mo-MSV RNA was generated by recombination between Mo-MLV and other genetic elements. We discuss whether all or only the MSV-specific sequences of the 30S Mo-MSV RNA function as sarcoma genes. Mo-MLV cDNA was hybridized about 45% by unfractionated Mo-MSV (MLV) RNA at RNA/DNA ratios of up to 10, about 50% by electrophoretically purified 30S Mo-MSV RNA at RNA/DNA ratios up to 500, but close to 100% by unfractionated Mo-MSV(MLV) RNA at RNA/DNA ratios over 900. This indicated that unfractionated RNA of our Mo-MSV(MLV) contained a complete complement of Mo-MLV, albeit at a low ratio.     

Infectivity and RNA Patterns as Functions of High- and Low-Dilution Passage of Murine Sarcoma-Leukemia Virus: Evidence for Autointerference Within an Oncornavirus Population

Heterogeneity of buoyant density and RNA content of virions of Moloney murine leukemia-sarcoma complex [MSV (MLV)] was the result of passage at low dilution. Heterogeneous stocks revealed two major RNA components in the population, with the smaller component, apparent mol wt 4 x 10(6) to 5 x 10(6), becoming predominant upon serial passage at low dilution. Concomitantly, infectivity titers of both MLV and MSV decreased upon serial passage at low dilution. MSV (MLV) passaged at high dilution retained high titers and a rather homogeneous high-molecular-weight RNA population characteristic of high-buoyant-density virions. Interference of both MLV and MSV replication was demonstrated by employing mixed inocula containing both low- and high-dilution passage stocks of MSV (MLV). In contrast to results with MSV (MLV), MLV freed of MSV by limit dilution did not show heterogeneity of buoyant density or of RNA when propagated at low dilution.     

Recombinational junctions of variants of Moloney murine sarcoma virus: generation and divergence of a mammalian transforming gene

Different variants of Moloney murine sarcoma virus (MSV) were examined by nucleotide sequencing to compare the junctions between the acquired cellular sequence, v-mos, and the adjacent virus-derived sequences. These variants included 124-MSV, m1-MSV, and HT1-MSV and also the purportedly independent isolate Gazdar MSV. These four strains have an identical 5' junction between the murine leukemia virus env gene and the v-mos gene. This junction lies within the sixth codon of the chimeric env-mos coding region that encodes the transforming gene product. In contrast, at the 3' junction between the v-mos gene and the murine leukemia virus env gene, the three variants examined here were all different. A small deletion was found in the COOH-terminal portion of the m1-MSV env-mos coding region, indicating that the COOH terminus of this transforming gene product must be different from that of 124-MSV or HT1-MSV. The data presented here are consistent with the thesis that a virus closely related to HT1-MSV was the primordial Moloney MSV, and that all other related strains evolved from it by deletion or rearrangement. The variability observed in the Moloney MSV family is discussed in terms of possible mechanisms for the initial capture of mos sequences by the parental retrovirus and also in comparison with other transforming retrovirus families, such as Abelson murine leukemia virus and Rous sarcoma virus.     

Core-Binding Factor Influences the Disease Specificity of Moloney Murine Leukemia Virus

The core site in the Moloney murine leukemia virus (Moloney MLV) enhancer was previously shown to be an important determinant of the T-cell disease specificity of the virus. Mutation of the core site resulted in a significant shift in disease specificity of the Moloney virus from T-cell leukemia to erythroleukemia. We and others have since determined that a protein that binds the core site, one of the core-binding factors (CBF) is highly expressed in thymus and is essential for hematopoiesis. Here we test the hypothesis that CBF plays a critical role in mediating pathogenesis of Moloney MLV in vivo. We measured the affinity of CBF for most core sites found in MLV enhancers, introduced sites with different affinities for CBF into the Moloney MLV genome, and determined the effects of these sites on viral pathogenesis. We found a correlation between CBF affinity and the latent period of disease onset, in that Moloney MLVs with high-affinity CBF binding sites induced leukemia following a shorter latent period than viruses with lower-affinity sites. The T-cell disease specificity of Moloney MLV also appeared to correlate with the affinity of CBF for its binding site. The data support a role for CBF in determining the pathogenic properties of Moloney MLV.     

Intracellular Viral RNA Species in Mouse Cells Nonproductively Transformed by the Murine Sarcoma Virus

The size and quantity of virus-specific RNA in five non-virus-producing mouse cells transformed by the Moloney isolate of murine sarcoma virus (MSV) was determined. Hybridization of RNA from transformed cells with the [(3)H]DNA product of the RNA-directed DNA polymerase of the murine sarcoma-leukemia virus was used to detect and quantitate virus-specific RNA. The amount of virus-specific RNA in non-virus-producing cells was less than one-sixth of that found in virus-producing cells. A striking correlation was found between the amount of intracellular virus-specific RNA and the degree of agglutination by conconavalin A previously reported for the four non-virus-producing NIH/3T3 cell lines (Salzberg and Green, 1974). A major RNA subunit sedimenting at 26 to 28S was detected in all five MSV-transformed non-virus-producing cells. This could represent the RNA genome of defective MSV.     

Size analysis and relationship of murine leukemia virus-specific mRNA's: evidence for transposition of sequences during synthesis and processing of subgenomic mRNA.

Virus-specific mRNA from purified polyribosomes of mouse cells infected with Moloney murine leukemia virus (M-MuLV) was analyzed by electrophoresis in agarose gels, followed by hybridization of gel slices with M-MuLV-specific complementary DNA (cDNA). The size resolution of the gels was better than that of sucrose gradients used in previous analyses, and two virus-specific mRNA's of 38S and 24S were detected. The 24S virus-specific mRNA is predominantly derived from the 3' half of the M-MuLV genome, since cDNAgag(pol) (complementary to the 5' half of the M-MuLV genome) could not efficiently anneal with this mRNA. However, sequences complementary to cDNA synthesized from the extreme 5' end of M-MuLV 38S RNA (cDNA 5') are present in the 24S virus-specific mRNA, since cDNA 5' (130 nucleotides) efficiently annealed with this mRNA. The annealing of cDNA 5' was not due to repetition of 5' terminal nucleotide sequences at the 3' end of M-MuLV 38S RNA, since smaller cDNA 5' molecules (60 to 70 nucleotides), which likely lack the terminal repetition, also efficiently annealed with the 24S mRNA. The sequences in 24S virus-specific mRNA recognized by cDNA 5' are not present in 3' fragments of virion RNA that are the same length. Therefore, it appears that RNA sequences from the extreme 5' end of the M-MuLV genome may be transposed to sequences from the 3' half of the M-MuLV 38S RNA during synthesis and processing of the 24S virus-specific mRNA. These results may indicate a phenomenon similar to the RNA splicing processes that occur during synthesis of adenovirus and papovavirus mRNA's.     

Biological and Serological Properties of Viral Particles from a Nonproducer Rat Neoplasm Induced by Murine Sarcoma Virus (Moloney)

The viral particles present in a nonproducer rat neoplasm induced by murine sarcoma virus (MSV) Moloney isolate, as detected by electron microscopy, were found to be biologically active on normal kidney cells of random-bred Osborne-Mendel rats. The virus is designated here as MSV (0). MSV (0) differs from other pseudotypes of MSV in its host range, antigenicity, and interference pattern.     

Heteroduplex analysis of the RNA of clone 3 Moloney murine sarcoma virus.

Heteroduplex analysis of the RNA isolated from purified virions of clone 3 Moloney murine sarcoma virus (M-MSV) hybridized to cDNA's from Moloney murine leukemia virus (M-MLV) and clone 124 M-MSV shows that the main physical component of clone 3 RNA is missing all or most of the 1.5-kilobase (kb) clone 124 M-MSV specific sequence denoted beta s (S. Hu et al. Cell 10:469--477, 1977). This sequence is either deleted in clone 3 RNA or substituted by a very short (0.3-kilobase) sequence. In other respects, clone 3 and clone 124 RNAs show the same heteroduplex structure relative to M-MLV. Since beta s is believed to contain the src gene(s) of clone 124 RNA, this result leaves as an unresolved question the nature of the src gene(s) of the clone 3 M-MSV RNA complex.     

DNA Polymerase of Murine Sarcoma-Leukemia Virus: Lack of Detectable RNase H and Low Activity With Viral RNA and Natural DNA Templates

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 <1:2,800 and 5,000. Based on the same activity with poly (A).oligo(dT) the activity of the murine DNA polymerase was 6 to 60 times lower than that of the avian enzyme with denatured salmon DNA template or with avian or murine viral RNA templates assayed under various conditions (native, heat-dissociated, with or without oligo(dT) and oligo(dC) and at different template enzyme ratios). The template activities of Ki-MSV(MLV) RNA and RSV RNA were enhanced uniformly by oligo(dT) but oligo(dC) was much less efficient in enhancing the activity of MSV(MLV) RNA than that of RSV RNA. It was concluded that the purified DNA polymerase of Ki-MSV(MLV) differs from that of Rous sarcoma virus in its lack of detectable RNase H and in its low capacity to transcribe viral RNA and denatured salmon DNA. Some aspects of these results are discussed.     

RNA-directed DNA synthesis in Moloney murine leukemia virus: interaction between the primer tRNA and the genome RNA.

Initiation of RNA-directed DNA synthesis in virions of Moloney murine leukemia virus requires a cellular tRNAPro as primer. The site(s) on the Moloney murine leukemia virus genome RNA at which functional primer molecules are bound and at which purified tRNAPro hybridizes has been located near (within 20%) the 5' end of the genome. A relatively stable duplex (temperature at which 50% dissociation has occurred, 76 degrees C) is formed between the amino acid acceptor stem of the tRNAPro and a complementary sequence in the Moloney murine leukemia virus 35S RNA. The interaction involves 19 base pairs, extending from the penultimate nucleotide at the 3' end of the tRNAPro but apparently not including the 3'-terminal adenosine residue. In most respects, the interaction between primer and template in Moloney murine leukemia virus parallels the situation in the avian leukosis-sarcoma viruses.     

Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.     

Transformation of Rat Liver Cells with Chicken Sarcoma Virus B77 and Murine Sarcoma Virus

Rat liver cells in vitro were transformed with chicken sarcoma virus B77, giving RL(B77) cells, and with murine sarcoma virus (Harvey), giving RL(MSV) cells. Rat liver cells transformed spontaneously in vitro were designated RL cells. In addition, the RL(MSV) cell line was adapted for growth in culture fluid containing 25 mug of 5-bromodeoxyuridine per ml. All cell lines were tumorigenic in 1-wk-old rats. The number of cells needed for induction of tumor growth was 1,000-fold higher in the case of RL(B77) cells in comparison with RL(MSV) cells and RL cells. No production of viral particles from any of the cell lines investigated was detected by plating concentrated supernatant fluid of the cultures on different secondary embryo cells with and without fusion by Sendai virus, by labeling with uridine-5-(3)H, or by assay for deoxyribonucleic acid polymerase activity. The viral genome was rescued by fusion of RL(B77) cells with chicken cells. Chicken sarcoma virus rescued from (RL(B77) cells differed in plating efficiency on duck cells from B77 virus rescued from transformed rat embryo cells. No virus was rescued after fusion of RL(MSV) and RL cells with mouse, rat, or chicken embryo cells. Infectious murine sarcoma virus can be induced by 5-bromodeoxyuridine from RL(MSV) cells.     

Effect of Helper Virus on the Number of Murine Sarcoma Virus DNA Copies in Infected Mammalian Cells

Cell lines of four mammalian species were each examined for the number of Moloney murine sarcoma virus (M-MSV) DNA copies in total cellular DNA after M-MSV transformation. Sarcoma-positive, leukemia-negative (S+L-) M-MSV-transformed cells were compared to M-MSV-transformed cells infected with a replicating leukemia virus. Both unfractionated M-MSV complementary DNA and complementary DNA representing the MSV-specific and the MSV-murine leukemia virus-common regions of the M-MSV genome were hybridized to total cellular DNA of various species. DNAs of mouse, cat, dog, and human S+L-cells contained from less than one to a few proviral M-MSV DNA copies per haploid genome. In contrast, helper virus-coinfected, M-MSV-producing cells of each species showed a 3- to 10-fold increase in M-MSV proviral DNA over that found in corresponding S+L- cells. MSV-specific and MSV-murine leukemia virus-common nucleotide sequences were each increased to a similar degree. A corresponding examination of cellular DNA of leukemia virus-infected normal or S+L- mammalian cells was performed to establish the resulting number of leukemia proviral DNA copies. The infection of normal or S+L- mammalian cells with several leukemia-type viruses that did not have nucleotide sequences closely related to the cell before infection resulted in the appearance of one to three corresponding leukemia proviral DNA copies.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.     

At least 104 nucleotides are transposed from the 5' terminus of the avian sarcoma virus genome to the 5' termini of smaller viral mRNAs.

Cells producing avian sarcoma virus (ASV) contain at least three virus-specific mRNAs, two of which are encoded within the 3' half of the viral genome. Each of these viral RNAs can hybridize with single-stranded DNA(cDNA5') that is complementary to a sequence of 101 nucleotides found at the 5' terminus of the ASV genome, but not within the 3' half of the genome. We proposed previously (Weiss, Varmus and Bishop, 1977) that this nucleotide sequence may be transposed to the 5' termini of viral mRNAs during the genesis of these RNAs. We now substantiate this proposal by reporting the isolation and chemical characterization of the nucleotide sequences complementary to cDNA5' in the genome and mRNAs of the Prague B strain of ASV. We isolated the three identified classes of ASVmRNA (38, 28 and 21S) by molecular hybridization; each class of RNA contained a "capped" oligonucleotide identical to that found at the 5' terminus of the ASV genome. When hybridized with cDNA5', each class of RNA gave rise to RNAase-resistant duplex hybrids that probably encompassed the full extent of cDNA5'. The molar yields of duplex conformed approximately to the number of virus-specific RNA molecules in the initial samples; hence most if not all of the molecules of virus-specific RNA could give rise to the duplexes. The duplexes prepared from the various RNAs all contained the capped oligonucleotide found at the 5' terminus of the viral genome and had identical "fingerprints" when analyzed by two-dimensional fractionation following hydrolysis with RNAase T1. In contrast, RNA representing the 3' half of the ASV genome did not form hybrids with cDNA5'. We conclude that a sequence of more than 100 nucleotides is transposed from the 5' end of the ASV genome to the 5' termini of smaller viral RNAs during the genesis of these RNAs. Transposition of nucleotide sequences during the production of mRNA has now been described for three families of animal viruses and may be a common feature of mRNA biogenesis in eucaryotic cells. The mechanism of transposition, however, and the function of the transposed sequences are not known.