Molecular evidence for a type C retrovirus etiology of the lymphoproliferative disease of turkeys.

Recently, we isolated from the blood of lymphoproliferative disease (LPD)-affected turkeys a type C retrovirus distinct from the avian leukosis-sarcoma virus complex and the reticuloendotheliosis virus group. We present molecular evidence for the implication of this virus in the LPD of turkeys. Using complementary DNA of LPD viral RNA, we found that the LPD viral genome is specifically and efficiently transcribed (2,500 copies per cell) in LPD tumor cells. Moreover, the LPD tumor cells contained newly inserted LPD viral information (5 to 10 copies per haploid genome), which was not present before the infection. From the absence of LPD virus-specific sequences in the normal cell genome of turkeys, it was concluded that the LPD virus is not an endogenous virus of turkeys. DNA-DNA annealing experiments revealed that the degree of sequence homology between LPD viral complementary DNA and cellular DNA of turkeys was not higher than that between LPD viral complementary DNA and cellular DNA of other species, thus indicating that the virus does not originate from turkeys.     

Serological Analysis of the Deoxyribonucleic Acid Polymerase of Avian Oncornaviruses II. Comparison of Avian Deoxyribonucleic Acid Polymerases

Monospecific antiserum prepared against the isolated deoxyribonucleic acid (DNA) polymerase of avian myeloblastosis virus (AMV) neutralized the endogenous ribonucleic acid-instructed DNA polymerase activity of detergent-disrupted virus. The viral polymerase was serologically unrelated to the seven major structural polypeptides of AMV. Furthermore, the viral enzyme was distinguished from normal cellular DNA polymerases by serological criteria; thus, antiserum against the viral enzyme neutralized its homologous antigen but not normal cellular DNA polymerases. Neutralization by antibody of viral DNA polymerase activity was observed with all avian leukemia-sarcoma viruses tested, irrespective of viral antigenic subtype. The DNA polymerase activity of avian reticuloendotheliosis virus, and of a variety of mammalian oncornaviruses, was not neutralized by antisera against the AMV polymerase. Immunological analysis of the RSValpha(O) mutant, which is deficient in DNA polymerase activity, shows this mutant to lack demonstrable polymerase antigen. Viral polymerase was identified by immunofluorescence as a cytoplasmic constituent in virus-producing chicken cells; polymerase antigen was not detected in uninfected (gs(-)) chicken cells.     

Lack of Serological Relationship Among DNA Polymerases of Avian Leukosis-Sarcoma Viruses, Reticuloendotheliosis Viruses, and Chicken Cells

Antibodies against a large and a small DNA polymerase isolated from chicken embryos and against avian myeloblastosis virus DNA polymerase were used to study the serological relationships of the DNA polymerase activities of three avian systems with RNA and a DNA polymerase-avian leukosis-sarcoma viruses, reticuloendotheliosis viruses, and a fraction from uninfected chicken cells. The DNA polymerase activity of disrupted virions of all avian leukosis-sarcoma viruses tested was neutralized to the same extent by antibody against avian myeloblastosis virus DNA polymerase and was not neutralized by the antibodies against chicken cellular DNA polymerases. The viruses tested included induced leukosis viruses and Rous-associated virus-O. The DNA polymerase activity of disrupted virions of all of the reticuloendotheliosis viruses was not neutralized by any of the antibodies. The chicken endogenous RNA-directed DNA polymerase activity was neutralized partially or completely, in different experiments, by antibody against the small DNA polymerase isolated from chicken embryos, but was not neutralized by the other two antibodies.     

Radioimmunological comparison of the DNA polymerases of avian retroviruses.

125I-labeled DNA polymerases of avian myeloblastosis virus and spleen necrosis virus were used in a radioimmunological characterization of avian retrovirus DNA polymerases. It was shown that avian leukosis virus and reticuloendotheliosis virus DNA polymerases do not cross-react in radioimmunoassays. Within the avian leukosis virus species, species-specific and type-specific antigenic determinants of the DNA polymerase were defined. The previous finding of genus-specific antigenic determinants in avian myeloblastosis virus and Amherst pheasant virus DNA polymerases was confirmed and extended to members of all subgroups of avian leukosis virus. It was shown that there is little immunological variation between the DNA polymerases of the four members of the reticuloendotheliosis virus species. Particles with RNA-dependent DNA polymerase activity from the allantoic fluid of normal chicken eggs and from the medium of a goose cell culture did not compete for the antibodies directed against any of the sets of antigenic determinants defined in this study.     

Purification and properties of spleen necrosis virus DNA polymerase.

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.     

Separation of Reticuloendotheliosis Virus from Avian Tumor Viruses

Velocity sedimentation and isopycnic density gradient centrifugation indicate that reticuloendotheliosis virus has a different mass and buoyant density than members of the avian tumor virus group. The group-specific antigen of the avian tumor virus group was not detected in concentrated and purified reticuloendotheliosis virus preparations.     

Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed.     

Avian reticuloendotheliosis viruses: evolutionary linkage with mammalian type C retroviruses.

Reticuloendotheliosis viruses have been shown to be causative of tumors in a variety of avian species. The major structural protein of these non-genetically transmitted viruses is demonstrated to possess antigenic determinants common to those of all known mammalian type C viruses. These findings establish a mammalian origin for this oncogenic avian retrovirus group. None of the known mammalian type C virus groups demonstrated a closer immunological relationship to avian reticuloendotheliosis viruses. These results suggest that reticuloendotheliosis viruses have been non-genetically transmitted for a long period of evolution or that these viruses may have arisen by relatively recent infection of birds with an as yet undiscovered mammalian type C retrovirus.     

Lack of Sequence Homology Among RNAs of Avian Leukosis-Sarcoma Viruses, Reticuloendotheliosis Viruses, and Chicken Endogenous RNA-Directed DNA Polymerase Activity

The relatedness of the RNAs of the three avian systems, including six avian leukosis-sarcoma viruses, four reticuloendotheliosis viruses, and the microsome fraction of normal uninfected chicken embryo cells, containing RNA and a DNA polymerase have been studied by nucleic acid hybridization. All six avian leukosis-sarcoma viruses have closely related nucleotide sequences; and all four reticuloendotheliosis viruses have closely related nucleotide sequences. But, almost no similarities were detected between the RNAs of avian leukosis-sarcoma viruses and reticuloendotheliosis viruses. The RNA template of the endogenous RNA-directed DNA polymerase activity of normal uninfected chicken cells had no detectable relationship to RNAs of avian leukosis-sarcoma and reticuloendotheliosis viruses.     

RNA polymerase activity in purified virions of avian reticuloendotheliosis viruses.

An RNA polymerase activity that synthesizes a U-rich RNA hydrogen bonded to a large viral RNA molecule was found in the cores of virions of avian reticuloendotheliosis viruses (REV). The RNA polymerase activity was separable from the DNA polymerase activity of REV virions. The 5'-terminus of the newly synthesized RNA was A. In addition, a tRNA nucleotidyl transferase activity, which added -CpCpA ends to tRNA, appears to be present in the REV virions.     

Basolateral maturation of retroviruses in polarized epithelial cells.

We have investigated the maturation sites of avian and mammalian C-type retroviruses in polarized epithelial cells. Examination of thin sections of Madin Darby canine kidney cells infected with RD114 or avian reticuloendotheliosis virus revealed that these viruses mature from the basolateral membrane domains. Similar results were obtained with a continuous line of mouse mammary epithelial cells infected with Friend, Moloney, Rauscher, or Kirsten murine leukemia viruses, or Friend virus-related or Moloney virus-related mink cell focus-forming viruses. Immunofluorescence observations indicate that viral glycoproteins are inserted only at the basolateral membranes in these cells. Because of the availability of DNA and protein sequence data, and of molecularly cloned viruses, these virus systems offer advantages for molecular studies on directional transport of plasma membrane glycoproteins.     

The location of v-src in a retrovirus vector determines whether the virus is toxic or transforming.

We prepared infectious stocks of an avian retrovirus, a modified spleen necrosis virus, containing the herpes simplex virus type 1 thymidine kinase gene and the avian sarcoma virus v-src gene. Viruses were recovered after cotransfection of chicken cells with DNA of recombinants between cloned spleen necrosis virus thymidine kinase and v-src and with DNA of cloned reticuloendotheliosis virus strain A. When v-src was inserted near the 5'end of the viral genome, only low titers of recombinant virus were recovered. Most of the recovered viruses were smaller than expected and did not transform the morphology of rat or chicken cells. A very small amount of virus of the expected structure was recovered; this virus transformed rat cells and expressed v-src. Cotransfection data indicated that one reason we failed to recover a significant titer of recombinant virus is that efficient expression of v-src is acutely toxic to chicken and dog cells. Insertion of v-src near the 3' end of the viral genome, such that it was expressed at a lower level compared with the 5'-v-src-containing virus, yielded a higher titer of recombinant virus, and this virus was transforming. The differences in the recovery and transforming activity of these viruses indicate that the location of an oncogene in the viral genome is an important factor regulating the level of its expression and whether or not this expression is toxic or transforming to cells.     

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.     

High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures.

Several 50 to 70S tumor viral RNAs have previously been shown by electron microscopy to be dimers, with the two monomer subunits joined near their 5' ends. Five additional naturally occurring type C RNA tumor viruses have now been examined: AKR, and endogenous murine ecotropic virus; NZB, an endogenous murine xenotropic virus; and ecotropic and an amphotropic virus isolated from a wild mouse; and the avian reticuloendotheliosis virus (REV). All five 50 to 70S RNAs have similar 5'-to-5' dimer structures. Therefore, the observations support the hypothesis that the dimer linkage is a structural feature common to all type C mammalian viruses. REV is the first example of an avian virus with a clear 5'-to 5' dimer linkage. All of the mammalian viral RNAs, but not REV, showed symmetrically placed loops in each subunit of the dimer. Possible molecular structures and biological functions of the dimer linkages and loops are discussed.     

Reticuloendotheliosis Virus Nucleic Acid Sequences in Cellular DNA

Reticuloendotheliosis virus 60S RNA labeled with (125)I, or reticuloendotheliosis virus complementary DNA labeled with (3)H, were hybridized to DNAs from infected chicken and pheasant cells. Most of the sequences of the viral RNA were found in the infected cell DNAs. The reticuloendotheliosis viruses, therefore, replicate through a DNA intermediate. The same labeled nucleic acids were hybridized to DNA of uninfected chicken, pheasant, quail, turkey, and duck. About 10% of the sequences of reticuloendotheliosis virus RNA were present in the DNA of uninfected chicken, pheasant, quail, and turkey. None were detected in DNA of duck. The specificity of the hybridization was shown by competition between unlabeled and (125)I-labeled viral RNAs and by determination of melting temperatures. In contrast, (125)I-labeled RNA of Rous-associated virus-O, an avian leukosis-sarcoma virus, hybridized 55% to DNA of uninfected chicken, 20% to DNA of uninfected pheasant, 15% to DNA of uninfected quail, 10% to DNA of uninfected turkey, and less than 1% to DNA of uninfected duck.     

DNA Polymerase of Murine Sarcoma-Leukemia Virus: Lack of Detectable RNase H and Low Activity With Viral RNA and Natural DNA Templates

Kirsten murine sarcoma-leukemia virus (Ki-MSV[MLV]) was found to contain less RNase H per unit of viral DNA polymerase than avian Rous sarcoma virus (RSV). Upon purification by chromatography on Sephadex G-200 and subsequent glycerol gradient sedimentation the avian DNA polymerase was obtained in association with a constant amount of RNase H. By contrast, equally purified DNA polymerase of Ki-MSV(MLV) and Moloney [Mo-MSV(MLV)] lacked detectable RNase H if assayed with two homopolymer and phage fd DNA-RNA hybrids as substrates. On the basis of picomoles of nucleotides turned over, the ratio of RNase H to purified avian DNA polymerase was 1:20 and that of RNase H to purified murine DNA polymerase ranged between <1:2,800 and 5,000. Based on the same activity with poly (A).oligo(dT) the activity of the murine DNA polymerase was 6 to 60 times lower than that of the avian enzyme with denatured salmon DNA template or with avian or murine viral RNA templates assayed under various conditions (native, heat-dissociated, with or without oligo(dT) and oligo(dC) and at different template enzyme ratios). The template activities of Ki-MSV(MLV) RNA and RSV RNA were enhanced uniformly by oligo(dT) but oligo(dC) was much less efficient in enhancing the activity of MSV(MLV) RNA than that of RSV RNA. It was concluded that the purified DNA polymerase of Ki-MSV(MLV) differs from that of Rous sarcoma virus in its lack of detectable RNase H and in its low capacity to transcribe viral RNA and denatured salmon DNA. Some aspects of these results are discussed.     

Avian reticuloendotheliosis virus: characterization of genome structure by heteroduplex mapping

The genome structure of defective, oncogenic avian reticuloendotheliosis virus (REV) was studied by heteroduplex mapping between the full-length complementary DNA of the helper virus REV-T1 and the 30S REV RNA. The REV genome (5.5 kilobases) had a deletion of 3.69 kilobases in the gag-pol region, confirming the genetic defectiveness of REV. In addition, REV lacked the sequences corresponding to the env gene but contained, instead, a contiguous stretch (1.6 to 1.9 kilobases) of the specific sequences presumably related to viral oncogenicity. Unlike those of other avian acute leukemia viruses, the transformation-specific sequences of REV were not contiguous with the gag-pol deletion. Thus, REV has a genome structure similar to that of a defective mink cell focus-inducing virus or a defective murine sarcoma virus. An additional class of heteroduplex molecules containing the gag-pol deletion and two other smaller deletion loops was observed. These molecules probably represented recombinants between the oncogenic REV and its helper virus.     

Pheasant virus DNA polymerase is related to avian leukosis virus DNA polymerase at the active site.

The DNA polymerase from Amherst pheasant virus (APV), a member of the pheasant virus species of retroviruses, was compared to the DNA polymerases of avian leukosis viruses (ALV) and a reticuloendotheliosis virus (spleen necrosis virus (SNV)). Immunoglobulin inhibition tests and competition immunoassays showed that APV and ALV DNA polymerases are closely related at their active sites. The determinants common to their active sites are not shared by SNV DNA polymerase. Bu using a species-specific radioimmunoassay, it was shown that both APV and SNV DNA polymerases are grossly different from ALV DNA polymerase. The specificity of the relationship of the active sites of APV and ALV DNA polymerases was confirmed by a heterologous radioimmunoassay. Our data indicate that pheasant viruses are evolutionarily linked to ALV.