A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA.

We conducted a mutational analysis within the previously defined encapsidation sequence (E) for spleen necrosis virus (SNV), an avian retrovirus. We found that two regions are necessary for efficient SNV replication. The first region is a double hairpin structure as proposed by Konings et al. (1992, J. Virol., 66, 632-640); the second region is located downstream of the hairpins. We showed further that the double hairpin structure is required for efficient SNV RNA encapsidation. Our work is the first to demonstrate, via linker-scanning and site-directed mutagenesis, that a specific RNA secondary structure is required for the encapsidation of retroviral RNA. Analysis of a series of mutations within the E region indicates (i) that preserving the secondary structure of the two hairpins is important for efficient encapsidation and (ii) that the stem regions of the hairpins contain specific sequences critical for encapsidation. Within the hairpins, the presence of at least one of the two conserved GACG four-residue loops, but not the moderately conserved bulge sequence of the first hairpin, is crucial for function. The function of the hairpins is independent of the relative order of the two hairpins. However, the two hairpins are not redundant and are not functionally identical. Replacement of SNV double hairpin sequences with those of Moloney murine leukemia virus (M-MLV) has no detectable effect on the replication of SNV-based retrovirus vectors with reticuloendotheliosis virus strain A (REV-A) helper virus. Furthermore, replacement of the entire E sequence of SNV with that of Moloney murine sarcoma virus (M-MSV) and M-MLV results in retroviral vectors that replicate as well as SNV vectors with wild type SNV E. This result indicates that the encapsidation sequences of M-MSV/M-MLV and SNV are not virus specific and that, during packaging of SNV and MLV RNA with viral proteins from REV-A, the encapsidation sequences are recognized largely by their secondary or tertiary structures.     

Heteroduplex analysis of the sequence relations between the RNAs of mink cell focus-inducing and murine leukemia viruses.

The sequence relationships betwen AKR ecotropic virus and an AKR-derived "mink cell focus-inducing" (MCF) isolate (AKR MCF 247), between Moloney murine leukemia virus (M-MLV) and an M-MLV MCF isolate (M-MLV83), and between AKR and M-MLV were studied by electron microscopic heteroduplex analysis. The MCF-specific sequences were found to map from 1.95 kilobases (kb) to 2.75 kb (+/- 0.15 kb) from the 3' end of the RNAs for both MCF isolates. The major sequence nonhomology regions between AKR and M-MLV lie between 0.9 and 3.5 kb from the 3' end. However, the AKR and M-MLV sequences immediately adjacent to the 1.95- and 2.75-kb junctions with MCF-specific sequences are relatively similar in AKR and M-MLV. Our results suggest that the env gene of MLVs maps from 1 kb to 3 kb from the 3' end of the genomic RNA and that the carboxyl end of the glycoprotein of each MCF strain is similar (or identical) to that of its ecotropic parent.     

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.     

Two base changes restore infectivity to a noninfectious molecular clone of Moloney murine leukemia virus (pMLV-1).

The complete nucleotide sequence of a molecular clone of Moloney murine leukemia virus (pMLV-1) has previously been reported (Shinnick et al., Nature [London] 293:543-548, 1981). However, pMLV-1 does not generate infectious virus after transfection into cells (Berns et al., J. Virol. 36:254-263, 1980). The lesion in pMLV-1 has been localized by determining the biological activity of recombinants containing DNA from an infectious clone of Moloney murine leukemia virus (pMLV-48) and pMLV-1. Replacement of a 1.0-kilobase pair region which spans the gag-pol junction of pMLV-1 with the corresponding DNA fragment from the infectious clone restores its infectivity. Nucleotide sequence analysis of this fragment obtained from the infectious clone (pMLV-48) and pMLV-1 reveals two single base pair changes, one in the p30gag gene and the other in the 5' end of the pol gene. The mutation in the pol gene does not affect the production of infectious virus but renders them XC negative, whereas the mutation in the gag gene appears to be lethal. The complete nucleotide sequence of an infectious clone of Moloney murine leukemia virus can now be deduced.     

Sequence homology between Moloney murine sarcoma virus and Moloney leukemia virus RNA.

The Moloney murine sarcoma-leukemia virus [M-MSV (MuLV)], propagated at high multiplicity of infection (MOI), was demonstrated previously to contain a native genome mass of 4 X 10(6) daltons as contrasted to a mass of 7 X 10(6) daltons for Moloney murine leukemia virus (M-MuLV). The 4 X 10(6)-dalton classof RNA from M-MSV (MuLV) was examined for base sequence homology with DNA complementary to the 7 X 10(6)-dalton M-MuLV RNA genome. Approximately 86% of the M-MSV (MuLV) was protected from RNase digestion by hybridization, whereas 95% of M-MuLV was protected under identical conditions. These results indicate that the small RNA class of high-MOI M-MSV (MuLV) contains little (perhaps 10%) genetic information not present in M-MuLV. Virtually all of the 1.8 X 10(6)-dalton subunits of M-MSV (MuLV) RNA contained regions of poly(A) since 94% of the RNA bound to oligo(dT) cellulose in 0.5 M KCl. This suggests that the formation of the 1.8 X 10(6)-dalton subunits occurs before their packaging into virions and does not result from hydrolysis of intact 3.5 X 10(6)-dalton subunits by a virion-associated nuclease.     

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.     

Use of the mouse mammary tumor virus long terminal repeat to promote steroid-inducible expression of v-mos.

We used the mouse mammary tumor virus long terminal repeat to promote dexamethasone-regulated expression of the Moloney murine sarcoma virus (M-MSV) transforming gene, v-mos. A recombinant DNA vector containing the mouse mammary tumor virus long terminal repeat fused to the M-MSV 124 v-mos gene was cotransfected with a plasmid containing the herpes simplex virus thymidine kinase gene (tk) into 3T3TK- cells. Individual clones of cells which grew in hypoxanthine-aminopterin-thymidine medium were tested for dexamethasone-regulated expression of p37mos as well as several transformation-specific phenotypic parameters. In the absence of dexamethasone, the v-mos transfectants appeared morphologically similar to the control cells despite low basal levels of p37mos expression. Upon hormone treatment, the levels of p37mos increased 5- to 10-fold, coincident with morphological changes typical of M-MSV transformation of 3T3 cells. The ability to form foci in monolayers also correlated with p37mos induction. The extent of morphological changes varied in individual clones of cells with similar levels of induced p37mos. Although the induced levels of p37mos were comparable to those seen in stable M-MSV 124 virus-transformed NIH 3T3 cells, the transfectants were unable to grow in soft agar under conditions which support growth of the virus-transformed cells. Acute infection of the transfectants with M-MSV 124 virus, a situation which resulted in elevated levels of p37mos, allowed these cells to grow in soft agar. The results described in this paper suggest that different threshold levels of p37mos may be necessary for the expression of various parameters of the transformed phenotype and also that continued expression of p37mos is necessary for maintenance of the transformed state. However, it also appears that the sensitivity to given levels of p37mos varies among clonal cell lines.     

Molecularly cloned c-mos(rat) is biologically active.

A unique rat cellular gene, c-mos(rat), homologous to the transforming sequences, v-mos, of Moloney murine sarcoma virus (M-MSV) was detected by hybridization to a v-mos specific probe. The c-mos(rat) gene was cloned together with its flanking sequences in an 11-kbp EcoRI DNA fragment inserted in vector Charon 4A. Two probes were used to investigate the position and orientation of c-mos(rat) in the clone examined ( D3e ), namely pMSV -31 which contains the sequences specific for the transforming sequences of M-MSV and pCS-1 which harbors 0.5 kbp of 5'-terminal sequences of c-mos(mouse) as well as 0.7 kbp of its flanking sequences. After ligation of a restriction fragment of clone D3e containing c-mos(rat) to a fragment containing the long terminal repeat of M-MSV and transfection of the DNA onto rat cells, we detected foci of transformed cells, thus showing that c-mos(rat) is biologically active. Using DNA framents derived from clone D3e , we studied the conservation of c-mos and of its flanking sequences in several species. c-mos(rat) as well as some of its flanking sequences appeared to be highly conserved in the species studied.     

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.     

Nucleotide sequence of AKV murine leukemia virus.

AKV is an endogenous, ecotropic murine leukemia virus that serves as one of the parents of the recombinant; oncogenic mink cell focus-forming viruses that arise in preleukemic AKR mice. I report the 8,374-nucleotide-long sequence of AKV, as determined from the infectious molecular clone AKR-623. The 5'-leader sequence of AKV extends to nucleotide 639, after which lies a long open reading frame encoding the gag and pol gene products. The reading frame is interrupted by a single amber codon separating the gag and pol genes. The pol gene overlaps the env gene within the 3' region of the AKV genome. The nucleotide sequence of the 5' region of AKV reveals the following features. (i) The 5'-leader sequence lacks any AUG codon to initiate translation of gPr80gag, suggesting that gPr80gag is not required for the replication of AKV. (ii) A short portion of the leader region diverges in sequence from the closely related Moloney murine leukemia virus and appears to be related to a sequence highly repeated in eucaryotic genomes. (iii) As in Moloney murine leukemia virus, there is a potential RNA secondary structure flanking the amber codon that separates the gag and pol genes. This structure might function as a regulatory protein binding site that controls the relative levels of synthesis of the gag and pol precursors. The nucleotide sequence of the 3' region of AKV is compared with sequences reported previously from both infectious and noninfectious molecular clones of AKV.     

Characterization of Gazdar murine sarcoma virus by nucleic acid hybridization and analysis of viral expression in cells.

Gazdar murine sarcoma virus (Gz-MSV) and Moloney murine sarcoma virus (M-MSV) are closely related. The complete M-MSV-specific nucleic acid sequences constituted a major portion of Gz-MSV-specific sequences. The MSV-specific sequences in both Gz-MSV and M-MSV genomes shared homology with hamster leukemia virus nucleic acid sequences. Both rat cells (S+L+) and hamster (S+L-) cells expressed two viral proteins of 68,000 and 70,000 daltons. These proteins were immunologically related to p60 purified from m1 virions of M-MSV.     

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.     

Construction of a mammalian transducing vector from the genome of Moloney murine leukemia virus.

A 0.9-kilobase DNA fragment from the genome of Moloney murine leukemia virus, including the viral long terminal repeat, was covalently linked to the herpes simplex virus I thymidine kinase (tk) gene whose promoter was previously removed. The hybrid DNA structure was introduced into the chromosome of tk- mouse cells at single copy numbers, via transfection procedures. Cells expressing the newly introduced tk gene were identified by the HAT selection procedure and analyzed for tk- and moloney murine leukemia virus-specific DNA and RNA sequences by blot hybridization procedures. Expression of the tk gene is dependent on function(s) provided in cis by the viral DNA fragment. Vectors derived from this region are termed rGag (rG) vectors.