Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA.

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was termed "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After cells acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplifications to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transformed, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.     

Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.     

In vitro methylation of specific regions of the cloned Moloney sarcoma virus genome inhibits its transforming activity.

The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as methylation at HpaII sites. In addition, methylation at both HpaII and HhaI sites did not further reduce the transforming activity of the DNA. These results suggested that; whereas methylation of specific sites on the provirus may not be essential for inhibiting the transforming activity of MSV DNA, methylation of specific regions may be necessary. Thus, by cotransfection of plasmids containing only specific regions of the MSV provirus, it was determined that methylation of the v-mos gene was more inhibitory to transformation than methylation of the viral long terminal repeat.     

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.     

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.     

Structural organization and biological activity of molecular clones of the integrated genome of a BALB/c mouse sarcoma virus.

BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin. The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda. In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol. Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection. A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome. Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA. A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA. A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs. These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.     

Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.

The nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed.     

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.     

Analysis of recombinant DNA clones of the endogenous BALB/c murine leukemia virus WN1802N: variation in long terminal repeat length

We analyzed 15 recombinant DNA clones of the unintegrated closed circular DNA intermediate of the BALB/c endogenous ecotropic murine leukemia virus WN1802N. Thirteen of these clones had an insert which corresponded to the complete murine leukemia virus genome. Of these, six contained a single long terminal repeat (LTR) and seven contained two LTRs. The viral genomes in nine clones had an LTR of 520 base pairs (bp), one had an LTR of 570 bp, three had an LTR of 600 bp, and one had an LTR of 670 bp. Restriction endonuclease analysis demonstrated that the size variability resides in the U3 region. Seven of eight clones which yielded infectious virus by DNA transfection had the 520-bp LTR, and the other had a 600-bp LTR. More detailed examination of plasmid subclones of three isolates with different-sized LTRs revealed that the approximate position which varies in the U3 region corresponds to the 72-bp repeat region of Moloney sarcoma virus. Possible consequences of these variations are discussed.