Sequence analysis of Gardner-Arnstein feline leukaemia virus envelope gene reveals common structural properties of mammalian retroviral envelope genes

We have sequenced the envelope (env) gene and most of the adjacent 3' long terminal repeat (LTR) of Gardner-Arnstein feline leukaemia virus subtype B. The LTR of this virus contains, at corresponding positions, all signal sequence elements known from other retroviral LTRs. The deduced amino acid sequence of the longest open reading frame was compared with env polypeptide sequences of several murine leukaemia viruses. This allowed us to predict the positions of both p12/15env and gp70 polypeptides as well as a hydrophobic leader polypeptide. The env polypeptides of the different viruses show long stretches of homology and similar hydrophilicity profiles in the p12env region and in the carboxy-terminal half of gp70 (constant region). The most extensive variations are confined to certain parts of the amino-terminal half of gp70 (differential region). In this region, however, feline leukaemia virus and murine mink cell focus forming viruses are still closely related. A correspondingly spaced pattern of identical, short amino acid sequences appears in three different parts of the env polyprotein, suggesting its evolution from a primordial env-related precursor by tandem duplications.     

Monoclonal antibody to spleen focus-forming virus-encoded gp52 provides a probe for the amino-terminal region of retroviral envelope proteins that confers dual tropism and xenotropism.

Monoclonal antibodies which recognize a region common to Friend spleen focus-forming virus encoded gp52 and Friend mink cell focus-inducing viral gp70 were isolated. One such antibody from hybridoma 7C10 was tested extensively in immune precipitation and was found to react with a determinant on envelope gp70s of all mink cell focus-inducing, xenotropic, and amphotropic mouse retroviruses tested, but not with envelope gp70s of ecotropic viruses, including Friend, Moloney, and AKR murine leukemia viruses. Monoclonal antibody from hybridoma 7C10 precipitated a 23,000-molecular-weight fragment, derived by V8 protease digestion of Friend mink cell focus-inducing gp70. This 23,000-molecular-weight peptide was determined to derive from the amino terminus of the molecule. These results correlate well with other genetic data which indicate that endogenously acquired sequences of mink cell focus-inducing viruses are found at the 5' end of the envelope gene.     

Generation of AKR mink cell focus-forming viruses: a conserved single-copy xenotrope-like provirus provides recombinant long terminal repeat sequences

AKV and AKR mink cell focus-forming virus-specific probes from the envelope and long terminal repeat (LTR) regions were prepared for study of the structure of recombinant proviruses in tumor tissues of AKR mice. The results showed that (i) all somatically acquired proviruses possessed, besides a recombinant gp70 gene, an altered U3 LTR; (ii) in a substantial portion of the somatically acquired AKR mink cell focus-forming proviruses, the LTR comprised sequences derived from the same xenotropic-like provirus; (iii) this U3 LTR donating parental provirus (Xeno-dL) was present only once per genome equivalent in several mouse strains; (iv) in the strains containing the Xeno-dL provirus, the provirus was present in the same chromosomal site; (v) restriction analysis of the Xeno-dL revealed that the mink cell focus-forming gp70 sequences were derived from a parental provirus, different from Xeno-dL. Therefore, at least two non-ecotropic parents participate in the generation of leukemogenic AKR mink cell focus-forming viruses: a xenotropic-like virus, Xeno-dL, donating U3 LTR sequences, and another xenotropic-like virus or viruses providing gp70 sequences.     

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.     

Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus

An infectious NZB xenotropic murine leukemia virus (MuLV) provirus (NZB was molecularly cloned from the Hirt supernatant of NZB-IU-6-infected mink cells, and the nucleotide sequence of its env gene and long terminal repeat (LTR) was determined. The partial nucleotide sequence previously reported for the env gene of NFS-Th-1 xenotropic proviral DNA (Repaske, et al., J. Virol. 46:204-211, 1983) is identical to that of the infectious NZB xenotropic MuLV DNA reported here. Alignment of nucleotide or deduced amino acid sequences, or both, of xenotropic, mink cell focus-forming, and ecotropic MuLV proviral DNAs in the env region identified sequence differences among the three host range classes of C-type MuLVs. Major differences were confined to the 5' half of env; a high degree of homology was found among the three classes of MuLVs in the 3' half of env. Alignment of the nucleotide sequence of the LTR of NZB xenotropic MuLV with those of the LTRs of NFS-Th-1 xenotropic, mink cell focus-forming, and ecotropic MuLVs revealed extensive homology between the LTRs of xenotropic and MCF247 MuLVs. An inserted 6-base-pair repeat 5' to the TATA box was a unique feature of both NZB and NFS-Th-1 xenotropic LTRs.     

Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA.

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the p15E peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses.     

At least four viral genes contribute to the leukemogenicity of murine retrovirus MCF 247 in AKR mice.

Nucleotide sequences encoding gp70, Prp15E, and the U3 region of the long terminal repeat (LTR) distinguish mink cell focus-forming (MCF) retroviruses that can induce leukemia in AKR mice from closely related MCF and ecotropic murine retroviruses that are nonleukemogenic in all inbred mouse strains tested (Lung et al., Cold Spring Harbor Symp. Quant. Biol. 44:1269-1274, 1979; Lung et al., J. Virol. 45:275-290, 1983). We used a set of recombinants constructed in vitro from molecular clones of leukemogenic MCF 247 and nonleukemogenic ecotropic Akv to separate and thereby directly test the role of these genetic elements in disease induction. Leukemogenicity tests of recombinants in AKR mice show that introduction of fragments containing either an MCF LTR or MCF gp70 coding sequences can confer only a very low incidence of disease induction on Akv virus, whereas an MCF type Prp15E alone is completely ineffective. Recombinants with an MCF 247 LTR in combination with MCF Prp15E are moderately oncogenic, whereas those with an MCF 247 LTR plus MCF gp70 coding segment are quite highly leukemogenic. Mice infected with the latter virus show a substantial increase in latent period of disease induction relative to MCF 247; this delay can be reduced when Prp15E, and hence the entire 3' half of the genome, is from MCF 247. Surprisingly, sequences in the 5' half of the genome can also contribute to disease induction. We found a good correlation between oncogenicity and recovery of MCF viruses from thymocytes of injected mice, with early recovery and high titers of MCF in the thymus being correlated with high oncogenicity. This correlation held for recombinants with either an MCF or ecotropic type gp70. Together, these results (i) demonstrate that at least four genes contribute to the oncogenicity of MCF viruses in AKR mice and (ii) suggest that recombinants with only some of the necessary MCF type genes induce leukemia because they recombine to generate complete MCF genomes. Although neither Akv nor MCF 247 is leukemogenic in NFS mice, recombinant viruses whose gp70 gene was derived from Akv but whose LTRs were derived from MCF 247 induced a low incidence of leukemia in this mouse strain.     

Nucleotide sequence of the env-specific segment of NFS-Th-1 xenotropic murine leukemia virus.

The sequence of 863 contiguous nucleotides encompassing portions of the pol and env genes of NFS-Th-1 xenotropic proviral DNA was determined. This region of the xenotropic murine leukemia virus genome contains and env-specific segment that hybridizes exclusively to xenotropic and mink cell focus-forming but not to ecotropic proviral DNAs (C. E. Buckler et al., J. Virol. 41:228-236, 1982). The unique xenotropic env segment contained several characteristic deletions and insertions relative to the analogous region in AKR and Moloney ecotropic murine leukemia viruses. Portions of an endogenous env segment cloned from a BALB/c mouse embryo gene library that had a restriction map and hybridization properties typical of xenotropic viruses (A. S. Khan et al., J. Virol. 44:625-636, 1982) were also sequenced. The sequence of the endogenous env gene was very similar to the comparable region of the NFS-Th-1 xenotropic virus containing the characteristic deletions and insertions previously observed and could represent a segment of an endogenous xenotropic provirus.     

Sequence comparisons of the anemia- and polycythemia-inducing strains of Friend spleen focus-forming virus.

A nucleotide sequence analysis carried out on the envelope gene of the anemia-inducing strain of the Friend spleen focus-forming virus (F-SFFVA) reveals that its product has some unique features in common with previously described polycythemia-inducing strains of F-SFFV (F-SFFVP). (i) It contains an amino terminus that is highly related to the gp70 of mink cell focus-inducing viruses, (ii) it is a fusion protein containing the amino terminus of gp70 and the carboxy terminus of p15E, and (iii) it lacks the R-peptide normally found at the carboxy end of the p15E region. Although the envelope genes of F-SFFVA and F-SFFVP are quite similar overall, they do show sequence variation, particularly at the 3' end in the p15E-related region. These variations may contribute to previously observed differences in the response of F-SFFVP- and F-SFFVA-infected erythroid cells to regulatory hormone or to differences in the way the envelope glycoproteins are processed. The long terminal repeat regions of F-SFFVA and the Lilly-Steeves strain of F-SFFVP were also sequenced and compared with each other and with a previously published sequence of another F-SFFVP long terminal repeat. The sequences were found to be reasonably similar to each other but different from their ecotropic parent, Friend murine leukemia virus, as a result of a deletion of one copy of the direct tandem repeat in the enhancer regions. The observation that all SFFVS have this common change in the long terminal repeat enhancer region raises the possibility that it is required for pathogenicity.     

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.     

Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA

The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. Despite their different pathogenicity, the nucleotide sequences of the env gene of Friend MCF virus and Moloney MCF virus were quite homologous, suggesting that the putative parent sequence for the generation of both MCF viruses and the recombinational mechanism for their generation might be the same. We compare the amino acid sequences in lymphoid leukemia-inducing ecotropic Moloney virus and Moloney MCF virus, and erythroblastic leukemia-inducing ecotropic Friend virus, Friend-MCF virus, and Friend spleen focus-forming virus. The Friend MCF virus long terminal repeat was found to be 550 base pairs long. This contained two copies of the 39-base-pair tandem repeat, whereas the spleen focus-forming virus genome contained a single copy of the same sequence.     

Cloning of endogenous murine leukemia virus-related sequences from chromosomal DNA of BALB/c and AKR/J mice: identification of an env progenitor of AKR-247 mink cell focus-forming proviral DNA

Recombinant phages containing murine leukemia virus (MuLV)-reactive DNA sequences were isolated after screening of a BALB/c mouse embryo DNA library and from shotgun cloning of EcoRI-restricted AKR/J mouse liver DNA. Twelve different clones were isolated which contained incomplete MuLV proviral DNA sequences extending various distances from either the 5' or 3' long terminal repeat (LTR) into the viral genome. Restriction maps indicated that the endogenous MuLV DNAs were related to xenotropic MuLVs, but they shared several unique restriction sites among themselves which were not present in known MuLV proviral DNAs. Analyses of internal restriction fragments of the endogenous LTRs suggested the existence of at least two size classes, both of which were larger than the LTRs of known ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. Five of the six cloned endogenous MuLV proviral DNAs which contained envelope (env) DNA sequences annealed to a xenotropic MuLV env-specific DNA probe; in addition, four of these five also hybridized to an ecotropic MuLV-specific env DNA probe. Cloned MCF 247 proviral DNA also contained such dual-reactive env sequences. One of the dual-reactive cloned endogenous MuLV DNAs contained an env region that was indistinguishable by AluI and HpaII digestion from the analogous segment in MCF 247 proviral DNA and may therefore represent a progenitor for the env gene of this recombinant MuLV. In addition, the endogenous MuLV DNAs were highly related by AluI cleavage to the Moloney MuLV provirus in the gag and pol regions.     

Pathogenicity of a Mutant Friend Spleen Focus-Forming Virus Encoding an Env-Like Membrane Glycoprotein (gp55) with Substitution by a Xenotropic Murine Leukemia Virus Env gp70 Sequence

Friend spleen focus-forming virus (F-SFFV) is a replication-defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)-like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus-inducing (MCF) murine leukemia virus (MuLV) Env gp70-derived sequence in its amino-terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino-terminal region. XE gp55 displayed almost the same biological activities as the wild-type gp55, excluding the above possibility.     

Genomes of murine leukemia viruses isolated from wild mice.

The genomes of murine leukemia viruses (MuLV) isolated from wild mice have been studied. Detailed restriction endonuclease maps of the 8.8-kilobase (kb) unintegrated linear viral DNAs were derived for five ecotropic and five amphotropic MuLV's from California field mice, for Friend MuLV, and for one ecotropic and one xenotropic MuLV from Mus musculus castaneus. In general, the California MuLV's were similar in their leftward 6 kb (corresponding to the leftward long terminal repeat [LTR], gag, and pol) and rightward 1 kb (7.8 to 8.8 kb, corresponding to p15E and the rightward LTR). For the region spanning 6.0 to 7.7 kb (which includes the sequences that encode gp70) the amphotropic MuLV's shared few enzyme sites with the ecotropic MuLV's, although the California ecotropic MuLV's were highly related to each other in this region, as were the amphotropic MuLV's. Cross-hybridization studies between amphotropic and California ecotropic MuLV DNAs indicated that they were not homologous in the region 6.3 to 7.6 kb; the California ecotropic viral DNAs cross-hybridized in this region to AKR ecotropic MuLV. When the California viral DNAs were compared with AKR ecotropic viral DNA, many differences in enzyme sites were noted throughout the genome. The U3 regions of the wild mouse LTRs showed partial homology to this region in AKR MuLV. The LTR of Moloney MuLV was highly related to that of the California MuLV's, whereas the LTR of Friend MuLV appeared to be a recombinant between the two types of LTRs. The M. musculus castaneus isolates were most closely related to ecotropic and xenotropic MuLV's isolated from inbred mice. One amphotropic MuLV DNA was cloned from supercoiled viral DNA at its unique EcoRI site in pBR322. Viral DNAs with one and two LTRs were isolated. After digestion with EcoRI, DNAs of both types were infectious. It is concluded that ecotropic and amphotropic MuLV's differ primarily in the region which encodes gp70.     

Ecotropic and mink cell focus-forming murine leukemia viruses integrate in mouse T, B, and non-T/non-B cell lymphoma DNA.

Structures of somatically acquired murine leukemia virus (MuLV) genomes present in the DNA of a large panel of MuLV-induced C57BL and BALB/c B and non-T/non-B cell lymphomas were compared with those present in MuLV-induced T-cell lymphomas induced in the same low-"spontaneous"-lymphoma-incidence mice. Analyses were performed with probes specific for the gp70, p15E, and U3-long terminal repeat (LTR) regions of ecotropic AKV MuLV and a mink cell focus-forming virus (MCF)-LTR probe annealing with U3-LTR sequences of a unique endogenous xenotropic MuLV, which also hybridizes with U3-LTR sequences of a substantial portion of somatically acquired MCF genomes in spontaneous AKR thymomas. The DNAs of both T- and B-cell tumors induced by neonatal inoculation with the highly oncogenic C57BL-derived MCF 1233 virus predominantly contain integrated MCF proviruses. In contrast, the DNAs of more slowly developing B and non-T/non-B cell lymphomas induced by poorly oncogenic ecotropic or MCF C57BL MuLV isolates mostly contain somatically acquired ecotropic MuLV genomes. Approximately 50% of the spontaneous C57BL lymphoma DNAs contain somatically acquired MuLV genomes. None of the integrated MuLV proviruses annealed with the MCF-LTR probe, which indicates a clear difference in LTR structure with a substantial portion of the somatically acquired MuLV genomes present in the DNA of spontaneous AKR thymomas. This study stresses a dominant role of MuLV with ecotropic gp70 and LTR sequences in the development of slowly arising MuLV-induced B and non-T/non-B cell lymphomas.     

Sequences responsible for erythroid and lymphoid leukemia in the long terminal repeats of Friend-mink cell focus-forming and Moloney murine leukemia viruses.

Despite the high degree of homology (91%) between the nucleotide sequences of the Friend-mink cell focus-forming (MCF) and the Moloney murine leukemia virus (MuLV) genomic long terminal repeats (LTRs), the pathogenicities determined by the LTR sequences of the two viruses are quite different. Friend-MCF MuLV is an erythroid leukemia virus, and Moloney MuLV is a lymphoid leukemia virus. To map the LTR sequences responsible for the different disease specificities, we constructed nine viruses with LTRs recombinant between the Friend-MCF and Moloney MuLVs. Analysis of the leukemia induced with the recombinant viruses showed that a 195-base-pair nucleotide sequence, including a 75-base-pair nucleotide Moloney enhancer, is responsible for the tissue-specific leukemogenicity of Moloney MuLV. However, not only the enhancer but also its downstream sequences appear to be necessary. The Moloney virus enhancer and its downstream sequence exerted a dominant effect over that of the Friend-MCF virus, but the enhancer sequence alone did not. The results that three of the nine recombinant viruses induced both erythroid and lymphoid leukemias supported the hypothesis that multiple viral genetic determinants control both the ability to cause leukemia and the type of leukemia induced.