Moloney Murine Leukemia Virus RNA Research Articles

An immature retroviral RNA genome resembles a kinetically trapped intermediate state.

Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. The structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.

Two distinct Moloney murine leukemia virus RNAs produced from a single locus dimerize at random

Two genetically distinct retroviral RNAs can be co-packaged if the RNAs are co-expressed in virion producing cells. For Moloney murine leukemia virus (MLV), co-packaged RNAs are not randomly selected from among all packaging-competent RNAs, but instead primarily associate as homodimers. Here, we tested the hypothesis that the distance between proviral templates might hinder RNA heterodimerization, thus generating the observed preferential homodimerization of co-expressed MLV RNAs. To do this, two genetically distinct RNAs were co-expressed from a single locus and the proportions of hetero- and homodimeric virion RNAs were determined. Unlike RNAs transcribed from two different templates, RNAs transcribed from a single locus dimerized at random. Additionally, in vitro transcription experiments suggested that MLV RNA dimerization can occur more efficiently for longer RNAs during transcription than post-synthesis. Together, these findings show that MLV RNA dimer-partner selection likely occurs either co-transcriptionally or within a pool of transcripts near the proviral template.

An additional dimer linkage structure in moloney murine leukemia virus RNA

We have identified an additional dimerization linkage structure in the genome of Moloney murine leukemia virus (MoMLV). Retroviral genomes have long been known to be linked at their 5′ ends to form dimers. In MoMLV, a hairpin loop functioning as a dimer linkage structure (DLS) has previously been identified at nucleotides 278–303. Here, we describe RNA dimers formed from sections of the MoMLV 5′ untranslated region that do not contain the previously described MoMLV DLS. These dimers exhibit the distinctive characteristics previously described for whole genome dimers. We have mapped this novel region to nucleotides 199–243. This sequence contains a stem-loop structure (nucleotides 204–227) much like the 278–303 region. We describe the chemical and thermal stability of dimers containing the 204–227 stem-loop as well as kinetics and salt-dependence of dimer formation. Our results show that dimerization of MoMLV RNA can be nucleated at multiple sites and suggest that the 5′ untranslated region may contain separately folding and dimerizing domains.

Modified nucleotides of tRNAPro restrict interactions in the binary Primer/Template complex of M-MuLV

In all retroviruses, reverse transcription is primed by a cellular tRNA, which is base-paired through its 3′-terminal 18 nucleotides to a complementary sequence on the viral RNA genome termed the primer binding site (PBS). Evidence for specific primer-template interactions in addition to this standard interaction has recently been demonstrated for several retroviruses. Here, we used chemical and enzymatic probing to investigate the interactions between Moloney murine leukemia virus (M-MuLV) RNA and its natural primer tRNAPro. The existence of extended interactions was further tested by comparing the viral RNA/tRNAProcomplex with simplified complexes in which viral RNA or tRNA were reduced to the 18 nt of the PBS or to the complementary tRNA sequence. These data, combined with computer modeling provide important clues on the secondary structure and three-dimensional folding of the M-MuLV RNA/tRNAProcomplex. In contrast with other retroviruses, we found that the interaction between tRNAProand the M-MuLV RNA template is restricted to the standard PBS interaction. In this binary complex, the viral RNA is highly constrained and the rest of tRNAProis rearranged, with the exception of the anticodon arm, leading to a very compact structure. Unexpectedly, when a synthetic tRNAProlacking the post-transcriptional modifications is substituted for the natural tRNAProprimer, the interactions between the primer and the viral RNA are extended. Hence, our data suggest that the post-transcriptional modifications of natural tRNAProprevent additional contacts between tRNAProand the U5 region of M-MuLV RNA.

Cis-active structural motifs involved in specific encapsidation of Moloney murine leukemia virus RNA.

We have analyzed the roles of RNA structural motifs located in the 5' part of the Moloney murine leukemia virus (M-MuLV) encapsidation domain (Psi region) with regard to their effects on viral replication. Four putative stem-loop structures between the 5' splice donor site and the gag initiation codon have been examined: stem structure A, corresponding to M-MuLV viral nucleotides 211 to 224; stem-loop B, nucleotides 278 to 303; stem-loop C, nucleotides 310 to 352; and stem-loop D, nucleotides 355 to 374. By measuring infectivities, encapsidation and splicing efficiencies, and endogenous reverse transcription levels of motif A, B, C, and D deletion mutants, we identified mutations which affect replication at the encapsidation step. In particular, deletion of all four motifs in a single mutant eliminated encapsidation of viral RNA, while deletion of individual elements moderately reduced the encapsidation efficiencies. Through analysis of different deletion combinations, we found that deletion of the first two motifs (A plus B) reduced both encapsidation and reverse transcription efficiencies, while deletion of the 3' motifs (C plus D) eliminated encapsidation. Interestingly, the C and D motifs both contain a GACG loop sequence and are highly conserved among murine type C retroviruses. Our results indicate that M-MuLV motifs C and D are necessary for efficient encapsidation, and the presence of at least one of these two stem-loops is crucial to encapsidation and virus replication.

A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.

Retroviral genomes consist of two identical RNA molecules joined noncovalently near their 5'-ends. Recently, two models have been proposed for RNA dimer formation on the basis of results obtained in vitro with human immunodeficiency virus type 1 RNA and Moloney murine leukemia virus RNA. It was first proposed that viral RNA dimerizes by forming an interstrand quadruple helix with purine tetrads. The second model postulates that RNA dimerization is initiated by a loop-loop interaction between the two RNA molecules. In order to better characterize the dimerization process of retroviral genomic RNA, we analyzed the in vitro dimerization of avian sarcoma-leukosis virus (ASLV) RNA using different transcripts. We determined the requirements for heterodimer formation, the thermal dissociation of RNA dimers, and the influence of antisense DNA oligonucleotides on dimer formation. Our results strongly suggest that purine tetrads are not involved in dimer formation. Data show that an autocomplementary sequence located upstream from the splice donor site and within a major packaging signal plays a crucial role in ASLV RNA dimer formation in vitro. This sequence is able to form a stem-loop structure, and phylogenetic analysis reveals that it is conserved in 28 different avian sarcoma and leukosis viruses. These results suggest that dimerization of ASLV RNA is initiated by a loop-loop interaction between two RNA molecules and provide an additional argument for the ubiquity of the dimerization process via loop-loop interaction.

A short autocomplementary sequence in the 5' leader region is responsible for dimerization of MoMuLV genomic RNA.

Previous work has shown that a region of Moloney murine leukemia virus (MoMuLV) RNA located between nucleotides 280 and 330 in the PSI region (nt 215-565) is implicated in the dimerization process. We show with a deletion from nucleotides 290-299 in PSI RNA transcripts and through an antisense oligonucleotide complementary to nucleotides 275-291 that the 283-298 region is involved in RNA dimer formation in vitro. In an attempt to further characterize the mechanism of dimer formation, a series of short RNA transcripts was synthesized which overlapps the PSI region of MoMuLV RNA. The dimerization of these RNAs is temperature dependent. The predicted secondary structure of the 278-303 region, as a function of temperature, reveals that this sequence is able to adopt two conformations: (1) the U288 AGCUA293 sequence in a loop; (2) part of the same nucleotides implicated in a stem. These results, together with thermodynamic analysis, strongly suggest that (1) the loop conformation of the UAGCUA sequence modulates the relative amount of RNA dimer and (2) a 16 bp long Watson-Crick base pairing is involved in RNA dimer formation. We propose that loop-loop recognition via the U288 AGCUA293 sequence leads to a stable structure induced by a stem-loop opening. Furthermore, our results do not support purine quartet formation as necessary for the dimerization of the 5' leader MoMuLV RNA.

Evidence that a central domain of nucleocapsid protein is required for RNA packaging in murine leukemia virus

We have analyzed RNA packaging by a series of mutants altered in the nucleocapsid (NC) protein of Moloney murine leukemia virus (Mo-MuLV). We found that mutants lacking residues 8 through 11 or 44 through 60 of NC package Mo-MuLV RNA with virtually the same efficiency as wild-type Mo-MuLV. In contrast, point mutants altered at the conserved cysteines in the cysteine array (residues 26 and 29) and a mutant lacking residues 16 through 23 packaged Mo-MuLV RNA with approximately 1% of the efficiency of wild-type Mo-MuLV. The deficiency in packaged RNA was observed not only in Northern (RNA) analysis but also in an RNA-PCR assay, which would detect degraded as well as intact RNA. One of the cysteine array mutants was also shown to be defective with respect to encapsidation of hygromycin phosphotransferase mRNA containing a Mo-MuLV packaging signal. We suggest that a central region of NC, consisting of the cysteine array and flanking basic residues, is required for RNA packaging in Mo-MuLV.

Spontaneous dimerization of retroviral MoMuLV RNA

The gerome of the Moloney murine leukemia virus (MoMuLV) is composed of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Dimerization sequences are located within the PSI encapsidation domain. We present here an overview of the work we have performed on spontaneous dimerization of a MoMuLV RNA fragment encompassing the PSI domain in order to understand the mechanism by which retroviral RNA dimerization takes place. We present kinetical, thermodynamical and conformational evidence which leads to the conclusion that the PSI domain is a structurally independent domain and that conformational changes are triggered by the dimerization process. We conclude that at least one particular region (nucleotides 278–309) of the RNA is directly involved in the process while the conformation of some other regions is changed probably because of a long-range effect.

Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging.

Site-directed mutagenesis has shown that the nucleocapsid (NC) protein of Rous sarcoma virus (RSV) is required for packaging and dimerization of viral RNA. However, it has not been possible to demonstrate, in vivo or in vitro, specific binding of viral RNA sequences by NC. To determine whether specific packaging of viral RNA is mediated by NC in vivo, we have constructed RSV mutants carrying sequences of Moloney murine leukemia virus (MoMuLV). Either the NC coding region alone, the psi RNA packaging sequence, or both the NC and psi sequences of MoMuLV were substituted for the corresponding regions of a full-length RSV clone to yield chimeric plasmid pAPrcMNC, pAPrc psi M, or pAPrcM psi M, respectively. In addition, a mutant of RSV in which the NC is completely deleted was tested as a control. Upon transfection, each of the chimeric mutants produced viral particles containing processed core proteins but were noninfectious. Thus, MoMuLV NC can replace RSV NC functionally in the assembly and release of mature virions but not in infectivity. Surprisingly, the full-deletion mutant showed a strong block in virus release, suggesting that NC is involved in virus assembly. Mutant PrcMNC packaged 50- to 100-fold less RSV RNA than did the wild type; in cotransfection experiments, MoMuLV RNA was preferentially packaged. This result suggests that the specific recognition of viral RNA during virus assembly involves, at least in part, the NC protein.

Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA

In Moloney murine leukemia virus, the encapsidation Psi element was shown to be necessary and sufficient to promote packaging of viral RNA, and to be required for dimerization. The conformation of the Psi domain (nucleotides 215 to 565) was investigated in solution by chemical probing. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate at cytosine N3 and adenosine N1, and with a carbodiimide derivative at guanosine N1 and uridine N3. Position N7 of adenine residues was probed with diethylpyrocarbonate. The analyses were conducted on in vitro transcribed fragments corresponding either to the isolated Psi domain or to the 5′-terminal 725 nucleotides. The RNA fragments were analyzed in their monomeric and dimeric forms. A secondary structure model was derived from probing data, computer prediction and sequence analysis of related murine retroviruses. One major result is that Psi forms an independent and highly structured domain. Dimerization induces an extensive reduction of reactivity in region 278 to 309 that can be interpreted as the result of intermolecular interactions and/or intramolecular conformational rearrangements. A second region (around position 215) was shown to display discrete reactivity changes upon dimerization. These two regions represent likely elements of dimerization. More unexpectedly, reactivity changes (essentially enhancement of reactivity) were also detected in another part of Psi (around position 480) not believed to contain elements of dimerization. These reactivity changes could be interpreted as dimerization-induced allosteric transitions.

Cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein.

To study the function of the retroviral nucleocapsid protein (NC), we have constructed point mutations in the gag gene of Moloney murine leukemia virus (MuLV) that affect a conserved cysteine-histidine motif of NC. The mutants were characterized biologically and biochemically. Cell lines producing the mutant virions were constructed in NIH 3T3 and rat2 cells, and the viral particles released by these cells were characterized for protein and RNA content. The results indicated that most mutations block replication and specifically inhibit the packaging of the MuLV genomic RNA. In some of the mutants, the packaging of the endogenous rat VL30 RNA was not affected as profoundly as was MuLV RNA. NC also seems to have another function distinct from dimer formation and packaging: one mutation reduced viral RNA packaging by only fivefold but completely abolished viral cDNA synthesis, suggesting a defect in reverse transcription.

Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus

A mutant of Moloney murine leukemia virus (M-MuLV), pMOV-ψ −, was constructed by deletion of about 350 nucleotides from an infectious proviral DNA clone between the putative env mRNA 5′ splice site and the AUG that initiates the coding sequence for Pr65 gag. Although the parent wild-type proviral clone, pMOV-ψ +, quickly causes a high level of reverse-transcriptase-containing virus particles to be released from transfected NIH/3T3 cells, transfection of pMOV-ψ − into these cells initially results in very little release. By 9 to 10 days after transfection, however, pMOV-ψ −-transfected cells produce infectious virus. Thus pMOV-ψ − has a defect that can be repaired in transfected NIH/3T3 cells, presumably by recombination with a sequence normally present in the cells. Cell lines with pMOV-ψ − stably integrated into chromosomal DNA produce reverse-transcriptase-containing particles that lack detectable M-MuLV RNA but the cells efficiently complement replication-defective, packagable retroviruses. Thus pMOV-ψ − has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production. The deletion in pMOV-ψ − appears to define a site required in cis for packaging of MuLV RNA into virions. Cell lines carrying pMOV-ψ − can be used to produce helper-free stocks of natural or synthetic defective retroviruses.

Secondary structural features in the 70S RNAs of Moloney murine leukemia and Rous sarcoma viruses as observed by electron microscopy.

The secondary structural features in the 70S RNAs of the Prague strain of avian Rous sarcoma virus, subgroup A (PR-RSV-A), and Moloney murine leukemia virus (M-MuLV) were compared by electron microscopy. The PR-RSV-A genome contained two subunits joined by a linkage structure as in the genomes of M-MuLV and other mammalian retroviruses. In both viral genomes, a highly reproducible hairpin occurred at about 70 nucleotides from the 5' end of each subunit and contained 320 +/- 8 nucleotides. The stable point of linkage between the subunits in both viral genomes involved fewer than 50 nucleotides and occurred at 466 +/- 9 nucleotides from the 5' end. This places the linkage about 350 nucleotides further toward the 3' end of the subunit than the binding site of primer tRNA. Another structural feature common to both genomes was a loop in each subunit. In M-MuLV, the loop contained 3.9 +/- 0.10 kilobases (kb) and occurred at a distance of 2.2 +/- 0.05 kb from the 5' end. In PR-RSV-A, the loop was smaller (2.3 +/- 0.10 kb) and further (3.3 +/- 0.10 kb) from the 5' end. When M-MuLV RNA was heated to 70, 85, or 90 degrees C and cooled, the hairpin consistently reformed at the 5' end. No other structures typical of the native molecules reappeared. In RNA samples heated to 70 degrees C, a new loop reproducibly occurred near the 5' end of each subunit, but this loop was not found in samples heated to higher temperatures. Based on all of these findings, we conclude that the genome of PR-RSV-A shares several features with M-MuLV and other mammalian retroviruses and that the primer tRNA molecules are not involved in the linkage of the two subunits in either genome. We also conclude that the dimer linkage and the loops in subunits are typical of the native molecules and that their formation requires a special environment.

Multiple integration sites for Moloney murine leukemia virus in productively infected mouse fibroblasts.

The integration sites for viral DNA in cells infected with Moloney murine leukemia virus (M-MuLV) were studied by restriction endonuclease cleavage of cellular DNA followed by electrophoresis in agarose gels, blot transfer to nitrocellulose, and detection by M-MuLV-related sequences by hybridization with high-specific-activity 32P-labeled M-MuLV complementary DNA. When EcoRI was used to cleave cellular DNA, numerous DNA fragments with sequence homology to M-MuLV were detected in uninfected mouse cell DNA. These endogenous sequences are mouse specific since they are not detectable in rat cell DNA, and are related to the 38S genomic RNA of M-MuLV. Infected cells contain additional M-MuLV-specific DNA fragments which are not detected in uninfected cells. Different patterns of M-MuLV-specific DNA fragments were detected in each cloned infected line examined. These data suggest the existence of multiple sites for integration of M-MuLV DNA in infected mouse fibroblasts. Cleavage of infected cell DNA with BamHI, which cleaves M-MuLV viral DNA at least twice, released the internal BamHI B fragment from each infected line, confirming the presence of integrated M-MuLV DNA sequences in each infected cell line which retain some features of the sequence organization of unintegrated M-MuLV DNA.

Translation of MuLV and MSV RNAs in nuclease-treated reticulocyte extracts: Enhancement of the gag-pol polypeptide with yeast suppressor tRNA

The virion RNAs from Moloney murine leukemia virus (MuLV) and Moloney murine sarcoma virus (MSV) were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The predominant polypeptides formed from 35S MuLV RNA were 78,000 and 65,000 daltons in molecular weight, and minor components with molecular weights of 180,000,110,000, 52,000 and 40,000 daltons were also observed. The 30S MSV RNA yielded two predominant polypeptides of 62,000 and 43,000 daltons, and minor components about 72,000, 40,000 and 18,000 daltons in molecular weight. The predominant polypeptides generated by both MuLV and MSV RNA were found to be precursors of the core proteins by immunoprecipitation with specific antisera. The 180,000 dalton molecular weight polypeptide encoded by MuLV RNA was immunoprecipitated both by antisera to the core protein (p30) and reverse transcriptase. The major products therefore appear to be Pr65 gag and Pr78 gag ; an important minor product is Pr180 gag-pol . Most of the products of <65,000 daltons synthesized in either system contained sequences from the core protein precursor, but the 43,000 and 18,000 dalton molecular weight polypeptides generated by MSV RNA did not precipitate with antisera to MuLV proteins. When purified yeast suppressor tRNA was added to the translation mixture directed by 35S MuLV RNA, the amount of the Pr78 gag was reduced, while the yield of the Pr180 gag-pol was enhanced. Amber suppressor tRNA was about 3 times as effective as ochre suppressor tRNA and nonsuppressor tRNA. This pattern of suppression was also seen for an established amber mutation (UAG) in the synthetase gene of Qβ (Qβ aml), suggesting that it is a UAG codon which terminates synthesis of Pr78 gag . In the MSV system, the amber suppressor tRNA, and to a lesser extent the ochre suppressor tRNA, markedly increased the synthesis of the 72,000 dalton molecular weight polypeptide with a slight reduction of the 62,000 dalton protein. Since read-through between the core protein and reverse transcriptase genes occurs to a low level both in vivo and in vitro and can be enhanced in vitro by amber suppressor tRNA, these results suggest that a suppression mechanism may control the relative amounts of core protein and reverse transcriptase synthesized from 35S mRNA. Such a mechanism might be used more generally by mRNAs from mammalian cells.

Relationship Between Moloney Murine Leukemia and Sarcoma Virus RNAs: Purification and Hybridization Map of Complementary DNAs from Defined Regions of Moloney Murine Sarcoma Virus 124

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3' end (cDNA 3') was prepared and shown to also be complementary to the 3'-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the "MSV-specific" portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3' end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5'-terminal two-thirds, as well as to the 3'-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5'-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3' portion of about 1,000 nucleotides, which is also present at the 3' terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3' terminus; and (iii) a second "common" region, again shared with MLV, which extends from 2,500 nucleotides to the 5' terminus. This second common region appears to be located in the 5' half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to (3)H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.

Rate of divergence of cellular sequences homologous to segments of Moloney sarcoma virus

The RNA genome of the Moloney isolate of murine sarcoma virus (M-MSV) consists of two parts--a sarcoma-specific region with no homology to known leukemia viral RNAs, and a shared region present also in Moloney murine leukemia virus RNA. Complementary DNA was isolated which was specific for each part of the M-MSV genome. The DNA of a number of mammalian species was examined for the presence of nucleotide sequences homologous with the two M-MSV regions. Both sets of viral sequences had homologous nucleotide sequences present in normal mouse cellular DNA. MSV-specific sequences found in mouse cellular DNA closely matched those nucleotide sequences found in M-MSV as seen by comparisons of thermal denaturation profiles. In all normal mouse cells tested, the cellular set of M-MSV-specific nucleotide sequences was present in DNA as one to a few copies per cell. The rate of base substitution of M-MSV nucleotide sequences was compared with the rate of evolution of both unique sequences and the hemoglobin gene of various species. Conservation of MSV-specific nucleotide sequences among species was similar to that of mouse globin gene(s) and greater than that of average unique cellular sequences. In contrast, cellular nucleotide sequences that are homologous to the M-MSV-murine leukemia virus "common" nucleotide region were present in multiple copies in mouse cells and were less well matched, as seen by reduced melting profiles of the hybrids. The cellular common nucleotide sequences diverged very rapidly during evolution, with a base substitution rate similar to that reported for some primate and avian endogenous virogenes. The observation that two sets of covalently linked viral sequences evolved at very different rates suggests that the origin of M-MSV may be different from endogenous helper viruses and that cellular sequences homologous to MSV-specific nucleotide sequences may be important to survival.

Methylation pattern of genomic RNA from Moloney murine leukemia virus.

32P- and methyl-3H-labeled 70S Moloney murine leukemia virus RNA was purified from virions produced in Moloney murine leukemia virus-infected mouse embryo cells. Primer-free RNA subunits obtained by heat treatment and zonal centrifugation were digested with RNase T2, and methylated oligonucleotides were chromatographed on DEAE-Sephadex in 7 M urea. Approximately one molecule of RNase T2-stable oligonucleotide (-5 charge) was isolated per subunit. Structural analysis indicated that the sequence of the oligonucleotide is m7GpppGmpCp. Analysis of the mononucleotide fraction isolated by DEAE-Sephadex chromatography of the RNase T2 digest identified 15 to 23 internal N6-methyladenylic acid molecules per subunit.