Evidence for genetic recombination between endogenous and exogenous mouse RNA type C viruses

Two viruses isolated following prolonged growth of serologically distinct mouse type C RNA viruses in human cells have previously been shown to have acquired common envelope properties distinct from those of either parental virus. Virus neutralization tests show that the viruses selected in human cells possess envelope antigens identical to those of endogenous mouse type C viruses of cells in which the parental viruses had been propagated. In contrast, the p12 polypeptide of each virus selected in human cells is antigenically indistinguishable from that of its respective parental virus and different from those of known endogenous mouse type C viruses. Molecular hybridization indicates significant differences in the genetic sequences of one virus and its parent, excluding the possibility that it arose from a point mutation. These findings indicate that the viruses selected in human cells represent genetic recombinants between exogenous and endogenous mouse type C viruses.     

Immunological relationships of OMC-1, an endogenous virus of owl monkeys, with mammalian and avian type C viruses

The relationships between OMC-1, an endogenous oncovirus of owl monkey, and representatives of the three oncoviral genera have been investigated by radioimmunological techniques. The major structural protein of OMC-1 was shown to share antigenic determinants with the corresponding proteins of certain type C viruses of rodent, feline, and cervine origin. It was not possible to demonstrate antigenic cross-reactivity between OMC-1 and endogenous type C viruses of baboons. These findings argue that OMC-1 and baboon endogenous viruses do not represent direct descendants of an ancestor virus that became integrated within primates prior to the divergence of New and Old World species. A close antigenic relationship was established between the major structural proteins of OMC-1, an endogenous virus of deer (deer kidney virus), and avian reticuloendotheliosis viruses. These findings establish OMC-1 and deer kidney virus in the evolutionary lineage that may have led to the generation of avian reticuloendotheliosis virus, a group of oncogenic viruses capable of crossing the interclass barrier between mammals and birds.     

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.     

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.     

Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses.

Western equine encephalomyelitis (WEE) virus (Togaviridae: Alphavirus) was shown previously to have arisen by recombination between eastern equine encephalomyelitis (EEE)- and Sindbis-like viruses (C. S. Hahn, S. Lustig, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 85:5997-6001, 1988). We have now examined the recombinational history and evolution of all viruses belonging to the WEE antigenic complex, including the Buggy Creek, Fort Morgan, Highlands J, Sindbis, Babanki, Ockelbo, Kyzylagach, Whataroa, and Aura viruses, using nucleotide sequences derived from representative strains. Two regions of the genome were examined: sequences of 477 nucleotides from the C terminus of the E1 envelope glycoprotein gene which in WEE virus was derived from the Sindbis-like virus parent, and 517 nucleotide sequences at the C terminus of the nsP4 gene which in WEE virus was derived from the EEE-like virus parent. Trees based on the E1 region indicated that all members of the WEE virus complex comprise a monophyletic group. Most closely related to WEE viruses are other New World members of the complex: the Highlands J, Buggy Creek, and Fort Morgan viruses. More distantly related WEE complex viruses included the Old World Sindbis, Babanki, Ockelbo, Kyzylagach, and Whataroa viruses, as well as the New World Aura virus. Detailed analyses of 38 strains of WEE virus revealed at least 4 major lineages; two were represented by isolates from Argentina, one was from Brazil, and a fourth contained isolates from many locations in South and North America as well as Cuba. Trees based on the nsP4 gene indicated that all New World WEE complex viruses except Aura virus are recombinants derived from EEE- and Sindbis-like virus ancestors. In contrast, the Old World members of the WEE complex, as well as Aura virus, did not appear to have recombinant genomes. Using an evolutionary rate estimate (2.8 x 10(-4) substitutions per nucleotide per year) obtained from E1-3' sequences of WEE viruses, we estimated that the recombination event occurred in the New World 1,300 to 1,900 years ago. This suggests that the alphaviruses originated in the New World a few thousand years ago.     

Molecular diversity among five different endogenous primate retroviruses.

Genetically transmitted retroviruses of Old and New World monkeys include type C viruses isolated from baboons (M7), macaque (MAC-1), and owl monkeys (OMC-1) and type D viruses from langurs (PO-1-Lu) and squirrel monkeys (SMRV, M534). Each of these isolates is unrelated to the others by nucleic acid hybridization criteria and contains a unique array of virion-associated proteins which can be resolved by agarose gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. The major structural protein of each virus has a distinct primary structure, as determined by two-dimensional tryptic peptide analysis, and is antigenically different from the others. The major virion phosphoproteins of endogenous primate type C viruses (pp15) are also different from those of type D viruses (pp13-pp14). Immunological and structural analyses show that the endogenous langur virus and the horizontally transmitted Mason-Pfizer virus of rhesus monkeys are closely related to one another, consistent with the sequence homology detected in their RNA genomes. Although certain radioimmunoassays detect interspecies antigenic determinants common to either the p30 or gp70 proteins of some of these viruses, no one assay has yet been designed which can detect all groups of endogenous primate retroviridae. The data lead to the conclusion that primates contain a minimum of three different sets of genetically transmitted type C and type D retroviral genes.     

Rat sequences of the Kirsten and Harvey murine sarcoma virus genomes: nature, origin, and expression in rat tumor RNA.

Two murine sarcoma viruses, the Kirsten and the Harvey, were isolated by passage of mouse type C leukemia viruses through rats. These sarcoma viruses have genomes containing portions of their parental type C mouse leukemia virus genomes, in stable association with specific rat cellular sequences that we find to be quite likely not those of a rat type C leukemia virus. To determine if these murine sarcoma viruses provide a model relevant to the events occurring in spontaneous tumors, we have hybridized DNA and RNA prepared from rat tumors and normal rat tissues to [3H]DNA prepared from the Kirsten murine sarcoma virus. We have also hybridized these rat tissue nucleic acids to [3H]DNA prepared from a respresentative endogenous rat type C leukemia virus, the WFU (Wistar-Furth). Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected in the DNA of both tumor and normal tissues, with no evidence of either gene amplification or additional sequences being present in tumor DNA. Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected at elevated concentrations in the RNA of many rat tumors and in specific groups of normal tissues.     

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.     

Skunks have gene sequences in their cellular DNA related to squirrel monkey retrovirus: transmission between species of a new world primate endogenous type D retrovirus.

The squirrel monkey ($\text{Saimiri}$,$\text{sciureus}$), a New World primate, contains multiple copies of endogenous type D retroviral gene sequences in the cellular DNA of all its tissues. Gene sequences partially homologous to these type D virus genes are also found in the cellular DNA of normal tissues of the New World carnivore, the skunk ($\text{Mephitis}$,$\text{mephitis}$ and $\text{Spilogale}$,$\text{putorius}$). We there-fore conclude that this class of viruses has, under natural conditions, been transmitted between the germ lines of these evolutionarily distant species. The example of interspecies transmission described here is the first that has been described among New World species and also the first that has been demonstrated for retroviruses other than type C viruses.     

Papio cynocephalus endogenous retrovirus among old world monkeys: evidence for coevolution and ancient cross-species transmissions

To study the evolutionary history of Papio cynocephalus endogenous retrovirus (PcEV), we analyzed the distribution and genetic characteristics of PcEV among 17 different species of primates. The viral pol-env and long terminal repeat and untranslated region (LTR-UTR) sequences could be recovered from all Old World species of the papionin tribe, which includes baboons, macaques, geladas, and mangabeys, but not from the New World monkeys and hominoids we tested. The Old World genera Cercopithecus and Miopithecus hosted either a PcEV variant with an incomplete genome or a virus with substantial mismatches in the LTR-UTR. A complete PcEV was found in the genome of Colobus guereza-but not in Colobus badius-with a copy number of 44 to 61 per diploid genome, comparable to that seen in papionins, and with a sequence most closely related to a virus of the papionin tribe. Analysis of evolutionary distances among PcEV sequences for synonymous and nonsynonymous sites indicated that purifying selection was operational during PcEV evolution. Phylogenetic analysis suggested that possibly two subtypes of PcEV entered the germ line of a common ancestor of the papionins and subsequently coevolved with their hosts. One strain of PcEV was apparently transmitted from a papionin ancestor to an ancestor of the central African lowland C. guereza.     

Identification of a 30S RNA with properties of a defective type C virus in murine cells.

A novel species of 30S RNA has been detected in a variety of mouse cell lines. The 30S RNA is specifically packaged by helper-independent type C viruses propagated in such cells. Nucleic acid hybridization detects no homology between the 30SRNA and the genomic RNA of helper-independent mouse type C viruses. The properties of the 30S RNA suggest that it is a defective endogenous mouse type C virus and that it is analogous to a previously described class of defective endogenous rat type C virus, which has been shown previously to be the progenitor of Kirsten and Harvey murine sarcoma viruses.     

Distribution of Mason-Pfizer Virus-Specific Sequences in the DNA of Primates

Iodinated Mason-Pfizer virus (MPV) 60-70S RNA has been used in molecular hybridization experiments to determine the distribution of MPV-specific proviral sequences in the DNAs of primates. Approximately 20% of the MPV genome is present as endogenous provirus in rhesus monkeys. Competitive hybridization experiments showed no homology between MPV 60-70S RNA and the 60-70S RNAs of M7, RD-114, and the simian sarcoma virus. No MPV-specific proviral sequences were detected in the DNAs of apparently normal tissues of various species of New World monkeys, apes, and humans. The part of the MPV genome that is endogenous to rhesus is also endogenous to the other species of Old World monkeys examined: baboon, African green, and patas. This was determined as a result of the following observations: (i) C(0)t(1/2) values and final extent of hybridization were the same for all four species. (ii) T(m) values of MPV 60-70S RNA and DNA of all four species were identical. (iii) The removal of MPV sequences endogenous to rhesus tissues by recycling against rhesus DNA resulted in the loss of any hybridizable MPV RNA to the DNAs of baboon, African green, and patas tissues. (iv) Mixing experiments of rhesus, African green, and baboon DNAs resulted in the same kinetics of hybridization as did rhesus DNA alone, when hybridized with MPV 60-70S RNA. These findings demonstrate that sequences that constitute an integral part of the MPV genome are conserved in the DNAs of several different species of Old World monkeys.     

Are type C viruses transforming agents?

Type C RNA viruses have been considered oncogenic because they are found associated with animal tumors and can induce cancers in several animal species. Those viruses that rapidly cause cancer appear to contain an oncogenic gene which resembles genetic sequences present in normal cells. This gene codes for a transforming protein which may be a normal cellular enzyme or a slightly altered cellular product. Its mechanism for transforming a cell is not yet known. Other oncogenic viruses, such as the chronic leukemia viruses, may not produce an oncogenic protein but may affect, by other means, specific target cells so they become malignant. Recent evidence now suggests that the majority of endogenous type C viruses are not transforming agents but inherited in the host to function in other biologic processes. These viruses do not contain transduced cellular genes which are responsible for cancer. Their role probably depends on their expression of other gene products which aid in normal development. These observations suggest that the ultimate control of human cancer may result from the identification of the oncogenic cellular-like genes transduced by some type C viruses even if a true human oncogenic virus is not isolated.     

New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor

Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position.