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Endogenous retroviruses
Cdl 5
in which the resolvase gene is inverted relative to its orientation in Tn3 and $, yet retains the same position adjacent to the transposase gene (Schmitt et al., In Molecular Biology, Pathogenicity and Ecology of Bacterial Plasmids, Plenum Press, pp. 359-370, 1981). It seems possible that the inversion was brought about by the action of resolvase at its normal res site and at a secondary (partially homologous) site that occurred fortuitously, close to the other end of the gene. Since (at least in Tn3 and yS) most of the specific elements of res lie between the normal crossover site and the start of tnpf?, such an inversion would be expected to retain the fully active res site close to this end of the
Cell, Vol. 32, 5-6,
January
Endogenous
1983,
Copyright
0 1983
gene; a prediction that can easily be tested. Although characterization of Tn3 and its transposition has progressed rapidly, many questions remain unanswered. The role of the transposase in the transpositional recombination has yet to be elucidated, and despite the variety of molecular models that have been proposed, there is as yet no information on how the transposon ends are joined to new target DNA. More progress has been made towards understanding the mechanism of site-specific cointegrate resolution, but again we are ignorant of the three-dimensional processes involved in formation of the resolved products.
by MIT
Retroviruses
Rudolf Jaenisch Heinrich-Pette-lnstitut fur Experimentelle Virologie und lmmunologie an der Universitat Hamburg Martinistrasse 52 2000 Hamburg 20, Federal Republic of Germany
The general structure of retroviruses has striking similarities with the structure of transposable elements found in procaryotes, yeast and Drosophila. These elements are flanked by long terminal repeats (LTRs) of several hundred base pairs, each of which is bounded by small inverted repeats and some of which have been shown to carry promoters of transcription. Integration of the elements generates a short direct duplication of host sequences at the insertion site. In Drosophila, extrachromosomal DNA copies of transposable elements (Flavell and Ish-Horowitz, Nature 292, 591-595, 1981) are found that are similar to DNA synthesized during retroviral replication. These structural and functional resemblances have led Temin (Cell 2 7, 599-600, 1980) to propose a common evolutionary origin of retroviruses and cellular transposable elements. However, evidence for intracellular transposition events of retroviral genomes without an RNA intermediate and an infectious cycle has not been obtained as yet. The genomes of endogenous retroviruses are found in multiple DNA copies in the chromosomes of many vertebrates. They are transmitted vertically as Mendelian genes and thus stand in contrast with exogenous viruses, which are transmitted horizontally from one animal to another. The endogenous viruses analyzed in greatest detail are those of the chicken and the mouse. The chicken genome carries at least 16 endogenous RAV-O-related proviral copies (ev-1 to ev-16) each at a unique chromosomal site (Astrin et al., CSHS 44, 1105-l 109, 1979). Some of these
proviral copies are able to specify the synthesis of infectious virus, whereas others represent defective genomes that may have lost part or all viral functions except a single LTR. Mouse strains exhibiting high tumor incidence were derived many years ago. Genetic evidence indicated that distinct Mendelian loci were involved in the synthesis of MuLV (murine leukemia virus, or C-type particle) in AKR mice or of MMTV (murine mammary tumor virus, or B-type particle) in GR mice. Development of malignancies was shown to be associated with unique proviral genomes and their expression. Biochemical analyses defined a number of distinct viral loci segregating in different high- and low-tumorincidence mouse strains (Jenkins et al., J. Virol. 43, 26-36, 1982). In addition to B- and C-type-related sequences, A-type and 30s VL (virus-like) sequences have been distinguished. The latter two classes do not form infectious virus particles, and although abundantly expressed during certain stages of development, their biological role remains obscure. Numerous experiments bear on the question of whether endogenous viruses have always coexisted with their host species as germline determinants. When germline proviruses of two species are more closely related to each other than are the cellular genes, one can suspect the possibility of horizontal transmission and insertion of virus into germline cells. Thus nucleic acid homology and immunological relatedness indicate that RD-114, one of the endogenous cat viruses, entered the cat germ line some 5-10 million years ago and probably originated from an endogenous primate virus by trans-species infection (Benveniste and Todaro, Nature 252, 456-459, 1974). The diversity of integration sites of murine Ctype (Jenkins et al., op. cit.) viruses suggests that germline infections have occurred repeatedly in mice. In fact, in the high-leukemia mouse strain AKR, new germline integrations are continuously accumulating,
Cell 6
with an average of one new virus acquired per 30 generations (Quint et al., J. Virol. 47, 901-908, 1982). The new proviral copies appear to result from exogenous infections, as no evidence for transposition events of retroviral genomes has been found. Infectious virus is produced in many tissues, notably the female reproductive tract of these viremic mice, which provides a route for infection of embryos and germline cells. This has been tested experimentally by exposing early mouse embryos to the exogenous Moloney leukemia virus. Genetic analysis of animals derived from infected embryos showed that proviruses were integrated in the DNA of germline cells with high frequencies. Thirteen different mouse substrains were derived, each transmitting the virus at a distinct Mendelian locus (Jaenisch et al., Cell 24, 519-529, 1981). Viral exposure of early embryos thus provides a possible route by which animals acquire new endogenous viruses. The expression of endogenous viruses appears to be under similar controls to those of developmentally regulated genes. Support for this comes from the correlation between chromatin structure, methylation and expression of endogenous retroviral genomes (Groudine et al., Nature 292, 31 l-31 7, 1981; Stuhlmann et al., Cell 26, 221-232, 1981). Furthermore, the chromosomal integration site of a given virus seems to influence the time in development and the tissue of virus activation (Jaenisch et al., op. cit.), suggesting that the activation of an endogenous virus in a given tissue may reflect transcriptional activity of the insertion site. This may explain the occasional expression of endogenous viruses during early development and in some tissues of the adult, which has led to the speculation that they have a role in cellular differentiation. At present there is no experimental evidence to support such a hypothesis. Individual mouse strains carrying different endogenous viruses express viral gene products in some of their tissues, each pattern of expression being idiosyncratic for a particular strain of mice. This is not compatible with a functional role of endogenous viruses in normal differentiation processes. Moreover, the success in breeding a healthy and fertile chicken lacking all endogenous viral sequences strongly argues against such a hypothesis (Astrin et al., Nature 282, 339-340, 1980). Instead, the occurrence of multiple copies of retrovirus-related sequences (estimates run as high as 0.3% of the total mouse genome) suggests that they behave like “parasitic” or “selfish” DNA. The detection of up to 100 proviral copies on the biologically largely inactive Y chromosome (Phillips et al., Nature 297, 241-243, 1982) is consistent with such a view. Endogenous viruses may, however, confer selective advantage on a host animal exposed to infectious virus particles. This is suggested by the unusual susceptibility of chickens free of functional endogenous viruses to the infection with exogenous retroviruses (Robinson et al., J. Virol. 40, 745-751,
1981). Retroviruses capable of insertion into many regions of the host genome are able to affect expression of cellular genes. This can occur either by enhancing gene transcription from a proximal position under the influence of the LTR or by physically disrupting cellular genes. Both phenomena have been observed: enhanced expression of cellular one genes by promoter insertion can lead to malignant transformation (Hayward et al., Nature 290, 475-479, 19811, and integration of Moloney leukemia virus into the src gene of an RSV-transformed cell line can affect src transcription, leading to reversion of the transformed phenotype (Varmus et al., Cell 25, 23-26, 1981). One may therefore suspect that, by similar mechanisms and depending on their site of integration, endogenous retroviruses may influence the function of cellular genes involved in embryogenesis and differentiation. This has indeed been observed in two instances. Jenkins et al. (Nature 293, 370-374, 1981) have demonstrated that a spontaneous coat-color mutation in mice is associated with insertion of a retroviral genome. More recently, a recessive-lethal mutation leading to early embryonic death has been induced experimentally by insertion of Moloney leukemia virus into the germ line of mice (Jaenisch et al., Cell 32, 209-216, 1983). The detailed mechanisms of viral integration are not known. Under the assumption that retroviruses integrate into active chromosomal regions, these elements may prove useful as insertion mutagens in identifying cellular genes involved in differentiation. Retroviruses have the ability to acquire and transduce foreign genes, probably by incorporation of mRNA into virus particles, reverse transcription and recombination. As a consequence, the cellular gene loses its introns. By this mechanism nontransforming retroviruses have acquired oncogenic potential by recombining with cellular genes, leading to the identification and isolation of proto-oncogenes that are present in all vertebrate cells. The remarkable mobility of eucaryotic genes and the discovery of intronless pseudogenes or “processed genes” (Hollis et al., Nature 296, 321-325, 1982) may indicate that transposition of genes to other chromosomal locations involves RNA’s copying into DNA. Recently, two retrovirus-like sequences have been found to flank a processed a-globin pseudogene (Lueders et al., Nature 295, 426-428, 1982). The LTRs flanking this gene, however, were inverted with respect to each other, making it difficult to derive a simple model of how the pseudogene could have evolved via a retroviral intermediate. Thus many questions about the role of retroviruses in the evolution of the eucaryotic genome remain open. It appears likely, however, that endogenous viruses will continue to provide the experimenter with powerful tools to study the organization and biology of animal cells.
in which the resolvase gene is inverted relative to its orientation in Tn3 and $, yet retains the same position adjacent to the transposase gene (Schmitt et al., In Molecular Biology, Pathogenicity and Ecology of Bacterial Plasmids, Plenum Press, pp. 359-370, 1981). It seems possible that the inversion was brought about by the action of resolvase at its normal res site and at a secondary (partially homologous) site that occurred fortuitously, close to the other end of the gene. Since (at least in Tn3 and yS) most of the specific elements of res lie between the normal crossover site and the start of tnpf?, such an inversion would be expected to retain the fully active res site close to this end of the
Cell, Vol. 32, 5-6,
January
Endogenous
1983,
Copyright
0 1983
gene; a prediction that can easily be tested. Although characterization of Tn3 and its transposition has progressed rapidly, many questions remain unanswered. The role of the transposase in the transpositional recombination has yet to be elucidated, and despite the variety of molecular models that have been proposed, there is as yet no information on how the transposon ends are joined to new target DNA. More progress has been made towards understanding the mechanism of site-specific cointegrate resolution, but again we are ignorant of the three-dimensional processes involved in formation of the resolved products.
by MIT
Retroviruses
Rudolf Jaenisch Heinrich-Pette-lnstitut fur Experimentelle Virologie und lmmunologie an der Universitat Hamburg Martinistrasse 52 2000 Hamburg 20, Federal Republic of Germany
The general structure of retroviruses has striking similarities with the structure of transposable elements found in procaryotes, yeast and Drosophila. These elements are flanked by long terminal repeats (LTRs) of several hundred base pairs, each of which is bounded by small inverted repeats and some of which have been shown to carry promoters of transcription. Integration of the elements generates a short direct duplication of host sequences at the insertion site. In Drosophila, extrachromosomal DNA copies of transposable elements (Flavell and Ish-Horowitz, Nature 292, 591-595, 1981) are found that are similar to DNA synthesized during retroviral replication. These structural and functional resemblances have led Temin (Cell 2 7, 599-600, 1980) to propose a common evolutionary origin of retroviruses and cellular transposable elements. However, evidence for intracellular transposition events of retroviral genomes without an RNA intermediate and an infectious cycle has not been obtained as yet. The genomes of endogenous retroviruses are found in multiple DNA copies in the chromosomes of many vertebrates. They are transmitted vertically as Mendelian genes and thus stand in contrast with exogenous viruses, which are transmitted horizontally from one animal to another. The endogenous viruses analyzed in greatest detail are those of the chicken and the mouse. The chicken genome carries at least 16 endogenous RAV-O-related proviral copies (ev-1 to ev-16) each at a unique chromosomal site (Astrin et al., CSHS 44, 1105-l 109, 1979). Some of these
proviral copies are able to specify the synthesis of infectious virus, whereas others represent defective genomes that may have lost part or all viral functions except a single LTR. Mouse strains exhibiting high tumor incidence were derived many years ago. Genetic evidence indicated that distinct Mendelian loci were involved in the synthesis of MuLV (murine leukemia virus, or C-type particle) in AKR mice or of MMTV (murine mammary tumor virus, or B-type particle) in GR mice. Development of malignancies was shown to be associated with unique proviral genomes and their expression. Biochemical analyses defined a number of distinct viral loci segregating in different high- and low-tumorincidence mouse strains (Jenkins et al., J. Virol. 43, 26-36, 1982). In addition to B- and C-type-related sequences, A-type and 30s VL (virus-like) sequences have been distinguished. The latter two classes do not form infectious virus particles, and although abundantly expressed during certain stages of development, their biological role remains obscure. Numerous experiments bear on the question of whether endogenous viruses have always coexisted with their host species as germline determinants. When germline proviruses of two species are more closely related to each other than are the cellular genes, one can suspect the possibility of horizontal transmission and insertion of virus into germline cells. Thus nucleic acid homology and immunological relatedness indicate that RD-114, one of the endogenous cat viruses, entered the cat germ line some 5-10 million years ago and probably originated from an endogenous primate virus by trans-species infection (Benveniste and Todaro, Nature 252, 456-459, 1974). The diversity of integration sites of murine Ctype (Jenkins et al., op. cit.) viruses suggests that germline infections have occurred repeatedly in mice. In fact, in the high-leukemia mouse strain AKR, new germline integrations are continuously accumulating,
Cell 6
with an average of one new virus acquired per 30 generations (Quint et al., J. Virol. 47, 901-908, 1982). The new proviral copies appear to result from exogenous infections, as no evidence for transposition events of retroviral genomes has been found. Infectious virus is produced in many tissues, notably the female reproductive tract of these viremic mice, which provides a route for infection of embryos and germline cells. This has been tested experimentally by exposing early mouse embryos to the exogenous Moloney leukemia virus. Genetic analysis of animals derived from infected embryos showed that proviruses were integrated in the DNA of germline cells with high frequencies. Thirteen different mouse substrains were derived, each transmitting the virus at a distinct Mendelian locus (Jaenisch et al., Cell 24, 519-529, 1981). Viral exposure of early embryos thus provides a possible route by which animals acquire new endogenous viruses. The expression of endogenous viruses appears to be under similar controls to those of developmentally regulated genes. Support for this comes from the correlation between chromatin structure, methylation and expression of endogenous retroviral genomes (Groudine et al., Nature 292, 31 l-31 7, 1981; Stuhlmann et al., Cell 26, 221-232, 1981). Furthermore, the chromosomal integration site of a given virus seems to influence the time in development and the tissue of virus activation (Jaenisch et al., op. cit.), suggesting that the activation of an endogenous virus in a given tissue may reflect transcriptional activity of the insertion site. This may explain the occasional expression of endogenous viruses during early development and in some tissues of the adult, which has led to the speculation that they have a role in cellular differentiation. At present there is no experimental evidence to support such a hypothesis. Individual mouse strains carrying different endogenous viruses express viral gene products in some of their tissues, each pattern of expression being idiosyncratic for a particular strain of mice. This is not compatible with a functional role of endogenous viruses in normal differentiation processes. Moreover, the success in breeding a healthy and fertile chicken lacking all endogenous viral sequences strongly argues against such a hypothesis (Astrin et al., Nature 282, 339-340, 1980). Instead, the occurrence of multiple copies of retrovirus-related sequences (estimates run as high as 0.3% of the total mouse genome) suggests that they behave like “parasitic” or “selfish” DNA. The detection of up to 100 proviral copies on the biologically largely inactive Y chromosome (Phillips et al., Nature 297, 241-243, 1982) is consistent with such a view. Endogenous viruses may, however, confer selective advantage on a host animal exposed to infectious virus particles. This is suggested by the unusual susceptibility of chickens free of functional endogenous viruses to the infection with exogenous retroviruses (Robinson et al., J. Virol. 40, 745-751,
1981). Retroviruses capable of insertion into many regions of the host genome are able to affect expression of cellular genes. This can occur either by enhancing gene transcription from a proximal position under the influence of the LTR or by physically disrupting cellular genes. Both phenomena have been observed: enhanced expression of cellular one genes by promoter insertion can lead to malignant transformation (Hayward et al., Nature 290, 475-479, 19811, and integration of Moloney leukemia virus into the src gene of an RSV-transformed cell line can affect src transcription, leading to reversion of the transformed phenotype (Varmus et al., Cell 25, 23-26, 1981). One may therefore suspect that, by similar mechanisms and depending on their site of integration, endogenous retroviruses may influence the function of cellular genes involved in embryogenesis and differentiation. This has indeed been observed in two instances. Jenkins et al. (Nature 293, 370-374, 1981) have demonstrated that a spontaneous coat-color mutation in mice is associated with insertion of a retroviral genome. More recently, a recessive-lethal mutation leading to early embryonic death has been induced experimentally by insertion of Moloney leukemia virus into the germ line of mice (Jaenisch et al., Cell 32, 209-216, 1983). The detailed mechanisms of viral integration are not known. Under the assumption that retroviruses integrate into active chromosomal regions, these elements may prove useful as insertion mutagens in identifying cellular genes involved in differentiation. Retroviruses have the ability to acquire and transduce foreign genes, probably by incorporation of mRNA into virus particles, reverse transcription and recombination. As a consequence, the cellular gene loses its introns. By this mechanism nontransforming retroviruses have acquired oncogenic potential by recombining with cellular genes, leading to the identification and isolation of proto-oncogenes that are present in all vertebrate cells. The remarkable mobility of eucaryotic genes and the discovery of intronless pseudogenes or “processed genes” (Hollis et al., Nature 296, 321-325, 1982) may indicate that transposition of genes to other chromosomal locations involves RNA’s copying into DNA. Recently, two retrovirus-like sequences have been found to flank a processed a-globin pseudogene (Lueders et al., Nature 295, 426-428, 1982). The LTRs flanking this gene, however, were inverted with respect to each other, making it difficult to derive a simple model of how the pseudogene could have evolved via a retroviral intermediate. Thus many questions about the role of retroviruses in the evolution of the eucaryotic genome remain open. It appears likely, however, that endogenous viruses will continue to provide the experimenter with powerful tools to study the organization and biology of animal cells.