Characterizatiion of rat genetic sequences of Kirsten sarcoma virus: distinct class of endogenous rat type C viral sequences.

The nucleic acid sequences found in DNA and RNA from rat cells which are homologous to Kirsten sarcoma virus have been characterized. The homologous sequences are present in multiple copies per diploid rat cellular genome in a variety of different rat cellular dna's. In certain cells that constitutively express only low levels of sequences homologous to Kirsten sarcoma virus, bromodeoxyuridine treatment leads to the expression of high levels of these sequences in RNA. Supernatants from cell lines producing the sequences homologous to Kirsten sarcoma virus contain high levels of these sequences which are purified to the same degree as the previously known rat type C viral nucleic acid sequences by type C particles being released from such cells. The results indicate that the sequences in rat cells homologous to Kisten sarcoma virus have three characteristics of known mammalian type C viruses, and suggest that at least part of Kirsten sarcoma virus rat-derived sequences represent a distinct class of endogenous rat type C virus that has no detectable homology to the other known class of endogenous rat type C virus.     

Distribution and Expression in Mammals of Genes Related to an Endogenous Type C RNA Virus of Odocoileus hemionus

An endogenous type C virus recently isolated from the Columbian black-tailed deer (Odocoileus hemionus) was used as a molecular probe to study the distribution of virus-related nucleotide sequences in cellular DNAs of mammalian species. By DNA-DNA hybridization, the most extensive homology was demonstrated between the viral complementary DNA and cellular DNA isolated from Odocoileus species. DNAs of representatives of other genera within the same family, Cervidae, were partially related to the virus, consistent with the phylogenetic relationship of these species to Odocoileus. O. hemionus viral sequences were also detected within cellular DNAs of members of a more distantly related artiodactyl family, Bovidae. These findings suggest the genetic transmission of type C viral genes within cervids and bovids for a period of at least 25 to 30 million years. There was no detectable nucleotide sequence homology between O. hemionus virus and representatives of other major groups of mammalian type C viruses. These results indicate that despite the known antigenic relatedness of mammalian type C viruses, the O. hemionus virus has diverged sufficiently to be considered the prototype of a separate group. By radioimmunological techniques, it was possible to detect and partially purify, from normal tissues of cervid species, antigens related to the major structural protein of the O. hemionus virus. The present findings, that O. hemionus virus has been genetically transmitted for millions of years and yet has maintained the ability to be expressed as infectious virus, argue for positive evolutionary selective pressures for the maintenance of type C viral genes.     

Two distinct endogenous type C viruses isolated from the asian rodent Mus cervicolor: conservation of virogene sequences in related rodent species.

The cocultivation of a lung cell line from the Southeast Asian mouse Mus cervicolor with cells from heterologous species has resulted in the isolation of two new distinct type C viruses. Both viruses are endogenous to M. cervicolor and are present in multiple copies in the cellular DNA of these mice. One of the viruses, designated M. cervicolor type CI, replicates readily in the SIRC rabbit cell line and is antigenically related to the infectious primate type C viruses isolated from a woolly monkey (simian sarcoma-associated virus) and gibbon apes (gibbon ape leukemia virus). This virus is also closely related by both immunological and nucleic acid hybridization criteria to a type C virus previously isolated from a second Asian murine species, Mus caroli. The isolation of the M. cervicolor type C I virus thus provides further evidence that the infectious primate type C viruses originated by trans-species infection of primates by an endogenous virus of mice. The second virus, designated M. cervicolor type C II, replicates well in various cell lines derived from the laboratory mouse Mus musculus. While antigenically related to type C viruses derived from M. musculus, the M. cervicolor type C II virus isolate can be readily distinguished from standard murine leukemia viruses. Both new type C viruses from M. cervicolor are unrelated to the previously described retrovirus (M432) isolated from the same Mus species. The DNA of M. cervicolor therefore contains multiple copies of at least three distinct classes of endogenous viral genes. An examination of the cellular DNA of other rodent species for nucleic acid sequences related to the genomes of both M. cervicolor type C I and II reveals that both viruses have been highly conserved evolutionarily, and that other species of rodents, such as laboratory mice and rats, contain endogenous virogenes related to those in the DNA of M. cervicolor.     

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.     

Genetic relationship between the Mus cervicolor M432 retrovirus and the Mus Musculus intracisternal type A particle

The genetic relationship between the retrovirus-like intracisternal type A particle (IAP) from Mus musculus and the novel retrovirus (M432) from M. cervicolor has been determined by heteroduplex and restriction endonuclease analyses of molecular clones of the respective genomes. We have found a major homology region (3.7 kilobase pairs) which probably begins near the 3' end of the M432 gag gene, spans the pol gene, and ends in the env gene. A second region (0.6 kilobase pairs) of weak homology was also observed adjacent to the 3' long terminal repeats of the respective genomes. The IAP genome is well conserved in the cellular DNA of all species of the genus Mus. In contrast, cellular DNA sequences related to the 5' end of the M432 genome, which shares no homology with the IAP genome, are found only in M. cervicolor and the closely related species M. cookii. These results suggest that the infectious M432 retroviral genome arose as a result of a recombinational event(s) between the IAP genome and another, as yet unidentified, class of retrovirus-related sequences or other cellular sequences.     

Endogenous mink (Mustela vison) type C virus isolated from sarcoma virus-transformed mink cells.

A previously described type virus stock (designated PP-1R), isolated by cocultivating baboon cells with mink cells transformed by Kirsten sarcoma virus (64J1), has been further cloned and characterized. End point-diluted stocks of PP-1R have been obtained that are free of focus-forming activity and lack both Kirsten sarcoma and primate type C viral sequences. Nucleic acid hybridization experiments show that the cloned virus (MiLV) is an endogenous, genetically transmitted virus of the mink (Mustela vison). MiLV replicates in canine, feline, and 64J1 mink cells but not in an untransformed mink cell line. Multiple viral gene copies can be detected in the DNA of normal mink cells in culture and in normal mink tissues; related endogenous viral genes are also detected in several related Mustela species. The virus codes for a p30 protein very closely related antigenically to that of feline leukemia virus but contains p15 and p12 proteins that are antigenically distinct. The mink cell line, Mv1Lu, and its Kirsten sarcoma-transformed derivatives, 64J1, express relatively low levels of type C viral RNA related to MiLV and normally do not produce detectable levels of MiLV p30 protein or complete, infectious viral particles. Infection of sarcoma virus-transformed mink cells with baboon type C virus, however, can augment the level of expression of endogenous mink viral RNA and can result in the synthesis and packaging of mink viral RNA and p30 antigen in extracellular virions. Since the Mv1Lu cell line and its tranformed derivatives have become widely used in studies of retroviruses, the possibility of activating endogenous mink viral genes should be considered by investigators working with these cells.     

Analysis of the virogenes related to the rhesus monkey endogenous type C retrovirus in monkeys and apes.

Molecular hybridization studies were carried out by using a [3H]complementary DNA (cDNA) probe to compare the endogenous type C retrovirus of rhesus monkeys (MMC-1) with other known retroviruses and related sequences in various primate DNAs. The genomic RNA of the endogenous type C retrovirus of stumptail monkeys (MAC-1) was found to be highly related to the MMC-1 cDNA probe, whereas the other retroviral RNAs tested showed no homology. Related sequences were found in Old World monkey DNAs and to a lesser extent in gorilla dn chimpanzee DNAs. No homology was detected between MMC-1 cDNA and DNA of gibbon, orangutan, or human origin. Restriction endonuclease analysis of genomic DNA indicated that many of the several hundred sequences related to MMC-1 in rhesus monkey DNA differed from that integrated into DNA of infected canine cells. Gorilla and chimpanzee DNAs contained a specific restriction endonuclease fragment of the MMC-1 genome.     

Biochemical characterization of the type C retrovirus associated with lymphoproliferative disease of turkeys.

Turkeys inoculated with spleen extracts from lymphoproliferative disease (LPD)-affected birds developed viremia, followed by typical LPD lesions. Electron microscopy and biochemical characterization established that the virus present in the blood of infected turkeys is a type C retrovirus. The viral particles possess a buoyant density of 1.17 g/ml in sucrose gradients; they contain high-molecular-weight RNA and an RNA-instructed DNA polymerase with efficient exogenous and endogenous activity. The LPD virus polymerase is preferentially activated by magnesium ions. Cross nucleic acid hybridization assays revealed no sequence homology between the viral genome of LPD and avian myeloblastosis virus or reticuloendotheliosis virus, thus indicating that the LPD virus belongs to a distinct group unrelated to the avian leukosis-sarcoma virus complex or to the reticuloendotheliosis virus group.     

Nucleotide sequence analysis of the linked left and right hand terminal regions of adenovirus type 5 DNA present in the transformed rat cell line 5RK20.

A peculiar phenomenon is observed in several adenovirus type 2 or 5 (Ad2 or Ad5) transformed cell lines: the right hand and left hand terminal regions of the viral genome present in the viral DNA insertions of these cell lines are found to be linked together. A large part of the viral DNA insertion present in the Ad5 transformed rat cell line 5RK20 has been cloned in the lambda vector Charon21A, including the segment containing the linked terminal regions. Sequence analysis of the linkage region showed a perfect homology with the Ad5 DNA sequence and a direct linkage of basepair (bp) 63 of the left hand end of the viral genome to bp 108 of the right hand end. No cellular or rearranged viral sequences were present. Our findings suggest that the joining of viral sequences into the cellular genome.     

Colobus type C virus: molecular cloning of unintegrated viral DNA and characterization of the endogenous viral genomes of Colobus.

The unintegrated viral DNA intermediates of colobus type C virus (CPC-1) were isolated from infected human cells that were permissive for viral growth. There were two major species of DNA, linear molecules with two copies of the long terminal repeat and relaxed circles containing only a single long terminal repeat. In addition, there was a minor species (approximately 10%) composed of relaxed circles with two copies of the long terminal repeat. A restriction endonuclease map of the unintegrated DNA was constructed. The three EcoRI fragments of circular CPC-1 DNA were cloned in the EcoRI site of lambda gtWES . lambda B and then subcloned in the EcoRI site of pBR322. Using these subgenomic fragments as probes, we have characterized the endogenous viral sequences found in colobus cellular DNA. They are not organized in tandem arrays, as is the case in some other gene families. The majority of sequences detected in cellular DNA have the same map as the CPC-1 unintegrated DNA at 17 of 18 restriction endonuclease sites. There are, however, other sequences that are present in multiple copies and do not correspond to the CPC-1 map. They do not contain CPC-1 sequences either in an altered form or fused to common nonviral sequences. Instead, they appear to be derived from a distinct family of sequences that is substantially diverged from the CPC-1 family. This second family of sequences, CPC-2, is also different from the sequences related to baboon endogenous type C virus that forms a third family of virus-related sequences in the colobus genome.     

Organization of type C viral DNA sequences endogenous to baboons: analysis with cloned viral DNA.

Unintegrated linear and circular forms of baboon endogenous type C virus M7 DNA were prepared from M7-infected cells by chromatography on hydroxyapatite columns, and the circular DNAs were purified in cesium chloride-ethidium bromide equilibrium density gradients. The circular DNAs were linearized by digestion with EcoRI, which had a unique site on the viral DNA. The linearized DNA was then inserted into lambda gtWES. lambda B at the EcoRI site and cloned in an approved EK2 host. Molecularly cloned full-length M7 DNA was restricted with BamHI, and the resulting five subgenomic fragments were then subcloned individually in plasmid pBR322. The organization and sites of integration of the approximately 100 copies of M7 DNA sequences endogenous to baboons were investigated by digesting the DNA with restriction enzymes and identifying the virus-specific fragments by hybridization to labeled probes made by using the molecularly cloned full-length and subgenomic fragments of the viral DNA. We found that most of the endogenous sequences had sizes and organizations similar to those of the unintegrated viral DNA and therefore approximately similar to the RNA of the infectious virus. A few of the multiple sequences had deletions in the 3' end (envelope region), and some of the sequences either lacked or contained modified BamHI restriction sites on the 5' end of the viral DNA. The endogenous viral DNA sequences were nontandem, uninterrupted, and colinear with the DNA of the infectious virus, and they were integrated at different sites in the baboon DNA, like the M7 proviral DNA sequences acquired upon infection.     

Primate retroviruses: envelope glycoproteins of endogenous type C and type D viruses possess common interspecies antigenic determinants.

The major 70,000- to 80,000-molecular-weight envelope glycoproteins of the squirrel monkey retrovirus, Mason-Pfizer monkey virus, and M7 baboon virus and the related endogenous feline virus, RD114, were isolated and immunologically characterized. Immunoprecipitation and competition immunoassay analysis revealed these viral envelope glycoproteins to possess several distinct classes of immunological determinants. These include species-specific determinants, group-specific antigenic determinants unique to endogenous primate type C viruses, and group-specific determinants for type D viruses such as Mason-Pfizer monkey virus and squirrel monkey retrovirus. In addition, a class of broadly reactive antigenic determinants shared by envelope glycoproteins of both type C viruses of the baboon/RD114 group and type D viruses of the Mason-Pfizer monkey virus/squirrel monkey virus group are described. Other mammalian oncornaviruses tested, including isolates of nonprimate origin and representative type B viruses, lacked these determinants. The demonstration of antigenic determinants specific to envelope glycoproteins of type C and type D primate viruses indicates either that these viruses are evolutionarily related or that genetic recombination occurred between their progenitors. Alternatively, endogenous type D oncornaviruses may be replication defective, and acquisition of endogenous type C viral genetic sequences coding for envelope glycoprotein determinants may be necessary for their isolation as infectious virus.