Two unrelated cell-derived sequences in the genome of avian leukemia and carcinoma inducing retrovirus MH2.

Molecularly cloned proviral DNA of avian replication-defective retrovirus Mill Hill No. 2 (MH2) was analyzed. The MH2 provirus measures 5.5 kb including two long terminal repeats (LTR), and contains a partial complement of the structural gene gag, 1.5 kb in size, near the 5' terminus, and a 1.3-kb segment of the v-myc transforming gene near the 3' terminus. These v-myc sequences are closely related to the v-myc transforming gene of avian acute leukemia virus MC29, and to the cellular chicken gene c-myc. The gag and myc domains on the MH2 provirus are separated by unique sequences, 1.3 kb in size and termed v-mil, which are unrelated to v-myc, or to other oncogenes or structural genes of the avian leukemia-sarcoma group of retroviruses. Normal chicken DNA contains sequences closely related to v-mil, termed c-mil. Analyses of chicken c-mil clones isolated from a recombinant DNA library of the chicken genome reveal that c-mil is a single genetic locus with a complex split gene structure. In the MH2 genome, v-mil is expressed via genome-sized mRNA as a gag-related hybrid protein, p100gag-mil, while v-myc is apparently expressed via subgenomic mRNA independently from major coding regions of structural genes. The presence in the MH2 genome of two unrelated cell-derived sequences and their independent expression may be significant for the oncogenic specificities of this virus.     

Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.     

A novel retroviruslike family in mouse DNA.

In the course of structural analysis of VL30 DNA elements, a recombinant retroviruslike element was encountered that contained non-VL30 long terminal repeats (LTRs) flanking internal VL30 sequences. With the aid of this novel LTR sequence probe, we cloned several DNA elements that were apparently members of a new retroviruslike family. A particular DNA element representative of this family (designated GLN) was characterized. It was approximately 8 kilobase pairs long and contained LTRs that are 430 base pairs long. It possessed an unusual primer-binding site sequence that corresponds to tRNAGln and a polypurine tract primer that is adjacent to the 3' LTR. The nucleotide sequences of the LTRs and their adjacent regions (which together housed all cis-acting retroviral functions) were different from those of known retroviruses and retroviruslike families. The comparison of three different GLN LTR sequences revealed a marked heterogeneity of U3 sequences relative to the homogeneity of R and U5 sequences. We estimate that approximately 20 to 50 copies of GLN elements are dispersed in all species of mice. GLN-related LTRs, however, are present in a much higher copy number (1,000 to 1,500 per genome). Nucleotide sequences that are more distantly related to GLN DNA are present in multiple copies in DNAs of other rodents but not in nonrodent genomes.     

Sequence of the Long Terminal Repeat and Adjacent Segments of the Endogenous Avian Virus Rous-Associated Virus 0

Rous-associated virus 0 (RAV-0), an endogenous chicken virus, does not cause disease when inoculated into susceptible domestic chickens. An infectious unintegrated circular RAV-0 DNA was molecularly cloned, and the sequence of the long terminal repeat (LTR) and adjacent segments was determined. The sequence of the LTR was found to be very similar to that of replication-defective endogenous virus EV-1. Like the EV-1 LTR, the RAV-0 LTR is smaller (278 base pairs instead of 330) than the LTRs of the oncogenic members of the avian sarcoma virus-avian leukosis virus group. There is, however, significant homology. The most striking differences are in the U(3) region of the LTR, and in this region there are a series of small segments present in the oncogenic viruses which are absent in RAV-0. These differences in the U(3) region of the LTR could account for the differences in the oncogenic potential of RAV-0 and the avian leukosis viruses. I also compared the regions adjacent to the RAV-0 LTR with the available avian sarcoma virus sequences. A segment of approximately 200 bases to the right of the LTR (toward gag) is almost identical in RAV-0 and the Prague C strain of Rous sarcoma virus. The segment of RAV-0 which lies between the end of the env gene and U(3) is approximately 190 bases in length. Essentially this entire segment is present between env and src in the Schmidt-Ruppin A strain of Rous sarcoma virus. Most of this segment is also present between env and src in Prague C; however, in Prague C there is an apparent deletion of 40 bases in the region adjacent to env. In Schmidt-Ruppin A, but not in Prague C, about half of this segment is also present between src and the LTR. This arrangement has implications for the mechanism by which src was acquired. The region which encoded the gp37 portion of env appears to be very similar in RAV-0 and the Rous sarcoma viruses. However, differences at the very end of env imply that the carboxy termini of RAV-0, Schmidt-Ruppin A, and Prague C gp37s are significantly different. The implications of these observations are considered.     

Nucleotide sequence of the intracisternal A-particle genome inserted 5'' to the interleukin-3 gene of the leukemia cell line WEHI-3B.

The nucleotide sequence of the intracisternal A-particle genome IAP-IL3 is presented. This IAP element was found to have inserted upstream of the promoter of the interleukin-3 gene of the leukemia cell line WEHI-3B. IAP-IL3 is 5095 bp in length, with identical long terminal repeats (LTRs) of 337 bp. The LTRs show many of the conserved sequence elements identified in other retroviruses. Comparison with other available sequences of IAP genomes indicates that IAP-IL3 is a deleted type I element. It carries a deletion covering the 3' end of the putative IAP gag gene and extending into the 5' end of the putative IAP pol gene. IAP-IL3 has extensive sequence homology with an IgE-binding factor cDNA and evidence is presented indicating that it was derived from a member of the mouse IAP sequence family. Comparison between the pol region of IAP-IL3 and other retroviruses suggests that IAP-IL3 is most closely related to type B and type D retroviruses.     

Apparent recombinants between virus-liKE (VL30) and murine leukemia virus-related sequences in mouse DNA.

VL30 elements are a dispersed multigene family that is ubiquitous in all murine cells. Despite not sharing nucleic acid sequence homology with natural retroviruses (exogenous or endogenous), VL30 elements are distinguished by several retrovirus-like features. By screening a mouse embryonic library, we have cloned DNA units that contain VL30 sequences linked to MuLV-related sequences. Using blot hybridization with the aid of specific subgenomic probes and heteroduplex analyses, we have established that the DNA element is composed of two VL30 long terminal repeat (LTR) units, a limited subset of VL30 information adjacent to both 5' and 3' LTRs, and an enclosure of MuLV-related information that shares homology primarily with MuLV gag and pol determinants (but lacks MuLV-related LTRs). This sequence arrangement is reciprocal in nature to the recombinations between MuLV and rat VL30 that generated the genomes of the Harvey and Kirsten strains of mouse sarcoma virus and most likely is the consequence of recombination between VL30 and MuLV-related elements and the subsequent deposition of the putative recombinant DNA in the mouse genome.     

CArG, CCAAT, and CCAAT-like protein binding sites in avian retrovirus long terminal repeat enhancers.

A strong enhancer element is located within the long terminal repeats (LTRs) of exogenous, oncogenic avian retroviruses, such as Rous sarcoma virus (RSV) and the avian leukosis viruses. The LTRs of a second class of avian retroviruses, the endogenous viruses (evs), lack detectable enhancer function, a property that correlates with major sequence differences between the LTRs of these two virus groups. Despite this lack of independent enhancer activity, we previously identified sequences in ev LTRs that were able to functionally replace essential enhancer domains from the RSV enhancer with which they share limited sequence similarity. To identify candidate enhancer domains in ev LTRs that are functionally equivalent to those in RSV LTRs, we analyzed and compared ev and RSV LTR-specific DNA-protein interactions. Using this approach, we identified two candidate enhancer domains and one deficiency in ev LTRs. One of the proposed ev enhancer domains was identified as a CArG box, a motif also found upstream of several muscle-specific genes, and as the core sequence of the c-fos serum response element. The RSV LTR contains two CArG motifs, one at a previously identified site and one identified in this report at the same relative location as the ev CArG motif. A second factor binding site that interacts with a heat-stable protein was also identified in ev LTRs and, contrary to previous suggestions, appears to be different from previously described exogenous virus enhancer binding proteins. Finally, a deficiency in factor binding was found within the one inverted CCAAT box in ev LTRs, affirming the importance of sequences that flank CCAAT motifs in factor binding and providing a candidate defect in the ev enhancer.     

Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.     

Influence of env and long terminal repeat sequences on the tissue tropism of avian leukosis viruses.

Adsorption and penetration of retroviruses into eucaryotic cells is mediated by retroviral envelope glycoproteins interacting with host receptors. Recombinant avian leukosis viruses (ALVs) differing only in envelope determinants that interact with host receptors for subgroup A or E ALVs have been found to have unexpectedly distinctive patterns of tissue-specific replication. Recombinants of both subgroups were highly expressed in bursal lymphocytes as well as in cultured chicken embryo fibroblasts. In contrast, the subgroup A but not subgroup E host range allowed high levels of expression in skeletal muscle, while subgroup E but not subgroup A envelope glycoproteins permitted efficient replication in the thymus. A subgroup B virus (RAV-2), like the subgroup E viruses, demonstrated a distinct bursal and thymic tropism, further supporting the theory that genes encoding receptors for subgroup B and E viruses are allelic. The source of long terminal repeats (LTRs) or adjacent sequences also influenced tissue-specific replication, with the LTRs from endogenous virus RAV-0 supporting efficient replication in the bursa and thymus but not in skeletal muscle. These results indicate that ALV env and LTR regions are responsible for unexpectedly distinctive tissue tropisms.