Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors.

We wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first we located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10(5) thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.     

Molecular cloning of the unintegrated squirrel monkey retrovirus genome: organization and distribution of related sequences in primate DNAs

The closed circular form of the endogenous squirrel monkey type D retrovirus (SMRV) was molecularly cloned in a bacteriophage vector. The restriction map of the biologically active clone was determined and found to be identical to that of the parental SMRV linear DNA except for the deletion of one long terminal repeat. Restriction enzyme analysis and Southern blotting indicated that the SMRV long terminal repeat was approximately 300 base pairs long. The SMRV restriction map was oriented to the viral RNA by using a gene-specific probe from baboon endogenous virus. Restriction enzyme digests of a variety of vertebrate DNAs were analyzed for DNA sequence homology with SMRV by using the cloned SMRV genome as a probe. Consistent with earlier studies, multiple copies of SMRV were detected in squirrel monkey DNA. Related fragments were also detected in the DNAs from other primate species, including humans.     

Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene.

To extend our understanding of the organization and expression of the mouse mammary tumor virus genome, we determined the nucleotide sequence of large regions of a cloned mouse mammary tumor virus strain C3H provirus that appears to be a DNA copy of env mRNA. In conjunction with analysis of several additional clones of integrated and unintegrated mouse mammary tumor virus DNAs, we came to the following conclusions: (i) the mRNA for env is generated by splicing mechanisms that recognize conventional eucaryotic signals at donor and acceptor sites with a leader of at least 289 bases in length; (ii) the first of three possible initiation codons for translation of env follows the splice junction by a single nucleotide and produces a signal peptide of 98 amino acids; (iii) the amino terminal sequence of the major virion glycoprotein gp52env is confirmed by nucleotide sequencing and is encoded by a sequence beginning 584 nucleotides from the 5' end of env mRNA; (iv) the final 17 amino acids at the carboxyl terminus of the primary product of env are encoded within the long terminal repeat by the 51 bases at the 5' end of the U3 domain; and (v) bases 2 through 4 at the 5' end of the long terminal repeat constitute an initiation codon that commences an open reading frame capable of directing the synthesis of a 36-kilodalton protein.     

A fps gene without gag gene sequences transforms cells in culture and induces tumors in chickens.

From molecularly cloned DNAs of Fujinami sarcoma virus (FSV) and the Schmidt-Ruppin-A strain of Rous sarcoma virus (SRA), viral DNA was constructed in which fps-specific sequences encoded in FSV replaced the src gene of SRA. A 3' fragment of FSV DNA, from an ATG methionine coding sequence 148 base pairs downstream from the gag-fps junction through the long terminal repeat, was joined to cloned SRA DNA at the translation start site for the src gene. The resultant DNA clone contained the splice acceptor site for src mRNA processing in SRA, but contained no src coding sequences from SRA nor any gag sequences from FSV. All genes for the replication of SRA were retained. Transfection of this cloned viral DNA genome into chicken embryo fibroblasts induced morphological transformation of the cells in culture. However, the morphology of the transformed cells was distinct from that observed in cells infected with wild-type FSV. The transformed cells produced a nondefective transforming virus called F36 which contained a hybrid FSV-SRA long terminal repeat. F36-infected cells produced a protein with the expected molecular weight of 91,000, which had an associated protein kinase activity and was immunoprecipitated by antibodies raised against fps gene determinants but not by antibodies raised against gag or src proteins. Injection of F36 virus into 8-day-old chicks produced tumors at the site of inoculation, detectable within 7 days. These results demonstrated that the gag portion of the gag-fps fusion protein of FSV is not required for transformation or tumorigenesis.     

The tandem direct repeats within the long terminal repeat of murine leukemia viruses are the primary determinant of their leukemogenic potential.

To map the viral sequences encoding the leukemogenic determinant(s) of nondefective murine leukemia viruses (MuLVs), we constructed chimeric viral genomes in vitro between cloned viral DNAs from the highly leukemogenic Gross passage A (Gross A) MuLV and from the related nonleukemogenic BALB/c N-tropic MuLV. Infectious chimeric MuLVs, recovered from murine cells microinjected with these DNAs, were inoculated into newborn mice to test the leukemogenic potential of these viruses. We found that the U3 long terminal repeat region from Gross A genomes was sufficient to confer an intermediate leukemogenic potential to chimeric MuLVs. Sequencing data indicated that the U3 tandem direct repeat was responsible for this effect. Adding most of the Gross A p15E-coding sequences to the Gross A U3 long terminal repeat enhanced the leukemogenic potential of chimeric viruses significantly. Adding a larger 3'-end env region (all p15E-coding sequences and 345 base pairs of the carboxy terminus of gp70) to the Gross A U3 long terminal repeat restored the full leukemogenic potential of Gross A MuLV. Chimeric viruses harboring only the Gross A 3'-end env region were, however, nonleukemogenic. Similar chimeric MuLVs, constructed with genomes from the parental weakly leukemogenic BALB/c B-tropic MuLVs and nonleukemogenic BALB/c N-tropic MuLVs, were also studied. Our data indicate that the U3 tandem direct repeat sequences appear to be necessary and sufficient to confer some leukemogenic potential to MuLV. However, env 3'-end sequences, mostly the p15E-encoding sequences, are required for the expression of fully leukemic phenotypes.     

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.     

Nucleotide sequence analysis of the long terminal repeat of avian myeloblastosis virus and adjacent host sequences.

The nucleotide sequence of the integrated avian myeloblastosis virus long terminal repeat has been determined. The sequence is 385 base pairs long and is present at both ends of the viral DNA. The cell-virus junctions at each end consist of a 6-base-pair direct repeat of cell DNA next to the inverted repeat of viral DNA. The long terminal repeat also contains promoter-like sequences, an mRNA capping site, and polyadenylation signals. Several features of this long terminal repeat suggest a structural and functional similarity with sequences of transposable and other genetic elements. Comparison of these sequences with long terminal repeats of other avian retroviruses indicates that there is a great variation in the 3' unique sequence (U3), whereas the 5' specific sequences (U5) and the R region are highly conserved.     

Molecular cloning of covalently closed circular DNA of bovine leukemia virus

The two species of covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES X lambda B. Of the nine independent recombinant lambda-bovine leukemia virus clones that were analyzed, three were derived from the small and six were derived from the large circular molecules carrying, respectively, one and two copies of the long terminal repeat sequences. Comprehensive restriction endonuclease mapping of the unintegrated bovine leukemia virus and the cloned DNA molecules showed that eight of the nine clones carried viral information without any detectable deletions or insertions of more than ca. 50 base pairs. One of the nine clones, which carries a retroviral insert with one copy of the long terminal repeat, had a deletion of ca. 150 base pairs.     

env Gene of Chicken RNA Tumor Viruses: Extent of Conservation in Cellular and Viral Genomes

The env gene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode env; our work exploited the availability of radioactive DNA (cDNA(gp)) complementary to most or all of env. env sequences were detectable in the DNAs of chickens which synthesized an env gene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no env gene product (chick helper factor negative). env sequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral env only in chicken DNA contrasts sharply with the demonstration that the transforming gene (src) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London) 260: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNA(gp) and chicken DNA and also between cDNA(gp) and RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNA(gp) did not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the env genes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin.     

Contribution of the gag and pol sequences to the leukemogenicity of Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV) is a highly leukemogenic replication-competent murine retrovirus. Both the F-MuLV envelope gene and the long terminal repeat (LTR) contribute to its pathogenic phenotype (A. Oliff, K. Signorelli, and L. Collins, J. Virol. 51:788-794, 1984). To determine whether the F-MuLV gag and pol genes also possess sequences that affect leukemogenicity, we generated recombinant viruses between the F-MuLV gag and pol genes and two other murine retroviruses, amphotrophic clone 4070 (Ampho) and Friend mink cell focus-inducing virus (Fr-MCF). The F-MuLV gag and pol genes were molecularly cloned on a 5.8-kilobase-pair DNA fragment. This 5.8-kilobase-pair F-MuLV DNA was joined to the Ampho envelope gene and LTR creating a hybrid viral DNA, F/A E+L. A second hybrid viral DNA, F/Fr ENV, was made by joining the 5.8-kilobase-pair F-MuLV DNA to the Fr-MCF envelope gene plus the F-MuLV LTR. F/A E+L and F/Fr ENV DNAs generated recombinant viruses upon transfection into NIH 3T3 cells. F/A E+L virus (F-MuLV gag and pol, Ampho env and LTR) induced leukemia in 20% of NIH Swiss mice after 6 months. Ampho-infected mice did not develop leukemia. F/Fr ENV virus (F-MuLV gag and pol, Fr-MCV env, F-MuLV LTR) induced leukemia in 46% of mice after 3 months. Recombinant viruses containing the Ampho gag and pol, Fr-MCF env, and F-MuLV LTR caused leukemia in 38% of mice after 6 months. We conclude that the F-MuLV gag and pol genes contain sequences that contribute to the pathogenicity of murine retroviruses. These sequences can convert a nonpathogenic virus into a leukemia-causing virus or increase the pathogenicity of viruses that are already leukemogenic.     

Sequences outside of the long terminal repeat determine the lymphomogenic potential of Rous-associated virus type 1.

Recombinant avian leukosis viruses have been constructed from the molecularly cloned DNAs of Rous-associated virus type 1 (RAV-1) and Rous-associated virus type 0(RAV-0). Virus encoded by the cloned RAV-1 DNA induced a high incidence of B-cell lymphoma and a moderate incidence of a variety of other neoplasms. Virus encoded by the cloned RAV-0 DNA did not cause disease. Virus recovered from DNA constructions that encoded the gag, pol, and 5' env sequences of RAV-0 and the 3' env and long terminal repeat sequences of RAV-1 did not cause a high incidence of lymphoma. Rather, these constructed viruses induced a low incidence of a variety of neoplasms. Virus recovered from reconstructed pRAV-1 DNA had the same disease potential as did virus recovered from the parental pRAV-1 DNA. These results indicate that the long terminal repeat sequences of RAV-1 do not confer the potential to induce a high incidence of B-cell lymphoma.     

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.     

Multiple viral determinants contribute to pathogenicity of the acutely lethal simian immunodeficiency virus SIVsmmPBj variant.

Simian immunodeficiency virus (SIV) induces an immunodeficiency syndrome similar to human AIDS. Although the disease course of SIV-induced immunodeficiency is generally measured in months to years, a disease syndrome that results in death in 5 to 14 days has been described in pig-tailed macaques infected with the SIVsmmPBj (PBj) strain. The purpose of this study was to derive an acutely lethal PBj molecular clone in order to study viral genes involved in pathogenesis. Six infectious molecular clones were generated; acutely fatal disease was induced by experimental inoculation of pig-tailed macaques with virus stocks derived from either of two clones, PBj6.6 or PBj14.6. Molecular chimeras were constructed by exchange of regions of the genome of PBj6.6 and a nonlethal, related clone, SIVsmH4. Only a chimera expressing the PBj genome under the control of a SIVsmH4 long terminal repeat induced death soon after inoculation. These studies suggest that multiple viral genes of PBj are critical for development of acute disease. More specifically, the env gene but not the long terminal repeat PBj was required for acute disease induction; however env must act in concert with another gene(s) of the PBj genome.