Endogenous retroviruses: still active after all these years?

Embedded in the genomes of all vertebrates are the proviral remnants of previous retroviral infections. Although the overwhelming majority has suffered inactivating mutations, current research suggests that members of one family of human retroelements may still be capable of movement.

Identification of the regions of Fv1 necessary for murine leukemia virus restriction.

The Fv1 gene restricts murine leukemia virus replication via an interaction with the viral capsid protein. To study this interaction, a number of mutations, including a series of N-terminal and C-terminal deletions, internal deletions, and a number of single-amino-acid substitutions, were introduced into the n and b alleles of the Fv1 gene and the effects of these changes on virus restriction were measured. A significant fraction of the Fv1 protein was not required for restriction; however, retention of an intact major homology region as well as of domains toward the N and C termini was essential. Binding specificity appeared to be a combinatorial property of a number of residues within the C-terminal portion of Fv1.

Multiple groups of novel retroviral genomes in pigs and related species.

In view of the concern over potential infection hazards in the use of porcine tissues and organs for xenotransplantation to humans, we investigated the diversity of porcine endogenous retrovirus (PERV) genomes in the DNA of domestic pigs and related species. In addition to the three known envelope subgroups of infectious gamma retroviruses (PERV-A, -B, and -C), classed together here as PERV group gamma 1, four novel groups of gamma retrovirus (gamma 2 to gamma 5) and four novel groups of beta retrovirus (beta 1 to beta 4) genomes were detected in pig DNA using generic and specific PCR primers. PCR quantification indicated that the retroviral genome copy number in the Landrace x Duroc F(1) hybrid pig ranged from 2 (beta 2 and gamma 5) to approximately 50 (gamma 1). The gamma 1, gamma 2, and beta 4 genomes were transcribed into RNA in adult kidney tissue. Apart from gamma 1, the retroviral genomes are not known to be infectious, and sequencing of a small number of amplified genome fragments revealed stop codons in putative open reading frames in several cases. Analysis of DNA from wild boar and other species of Old World pigs (Suidae) and New World peccaries (Tayassuidae) showed that one retrovirus group, beta 2, was common to all species tested, while the others were present among all Old World species but absent from New World species. The PERV-C subgroup of gamma1 genomes segregated among domestic pigs and were absent from two African species (red river hog and warthog). Thus domestic swine and their phylogenetic relatives harbor multiple groups of hitherto undescribed PERV genomes.

Endogenous retroviruses and the human germline.

The human genome is rife with the proviral remains of many ancient retroviruses. The past year has seen significant progress in understanding the structure, distribution and potential function of many of these elements. Although hypotheses concerning the potential effects of these elements are common, however, incisive experiments to test any functions remain much less so.

A conserved mechanism of retrovirus restriction in mammals.

The murine Fv1 gene restricts infection by N- or B-tropic murine leukemia viruses at a postentry, preintegration stage. The Fv1-sensitive viruses previously used for the study of Fv1 encode an ecotropic envelope gene and thus only infect rodent cells. Consequently, the study of Fv1 restriction has been carried out solely in mice and murine cell lines. By infection with retroviral vectors containing N- or B-tropic core and pantropic vesicular stomatitis virus-G envelope protein, we now demonstrate that cell lines derived from various mammalian species, including humans, have an Fv1-like retrovirus restriction function, preventing N-tropic vector infection. Like Fv1, restriction is directed at amino acid 110 of the viral capsid protein. In contrast to Fv1, the novel restriction is characterized by the absence of reverse-transcribed viral DNA. We speculate that these activities have been selected for by retroviral epidemics in the distant past.

Use of a transient assay for studying the genetic determinants of Fv1 restriction.

To probe the genetic determinants controlling the interaction between the retroviral restriction gene Fv1 and its murine leukemia virus target, we set out to develop rapid, transient assays for Fv1 function. Cells were transfected or transduced with Fv1 expression plasmids which can produce green fluorescent protein via an internal ribosome entry site positioned between the Fv1 and green fluorescent protein coding sequences. Fv1 function was then assessed by comparing virus replication in green fluorescent protein-positive and -negative cells, using retroviral vectors encoding a second fluorescent marker, yellow fluorescent protein, or beta-galactosidase. Using this assay, we could show that Fv1 specificities were not as absolute as previously thought, since the Fv1(b) allele was capable of interacting with "nonrestricted" B- and NB-tropic viruses and by shuffling the n- and b-alleles of Fv1, it was possible to generate a Fv1 molecule capable of restricting N-, B-, and NB-tropic viruses equally efficiently. Further, we could show that the presence of nonrestricting Fv1 in the same cell as restrictive Fv1 abrogates restriction, implying competition for binding to the retroviral target.

The hairless gene of the mouse: relationship of phenotypic effects with expression profile and genotype.

Various mutations of the hairless (hr) gene of mice result in hair loss and other integument defects. To examine the role of the hr gene in mouse development, the expression profile of hr has been determined by in situ hybridisation and correlated to the nature of genetic changes and morphological abnormalities in different mutant animals. Four variant alleles have been characterised at the molecular level. hr/hr mice produce reduced, but significant, levels of hr mRNA whereas other alleles contain mutations which would be expected to preclude the synthesis of functional product, demonstrating a correlation between allelic variation at the hr locus and phenotypic severity. hr expression was shown to be widespread and temporally regulated. It was identified in novel tissues such as cartilage, developing tooth, inner ear, retina, and colon as well as in skin and brain. Analysis of mice homozygous for the rhino allele of hairless revealed that, although no morphological defects were detectable in many tissues normally expressing hr, previously undescribed abnormalities were present in several tissues including inner ear, retina, and colon. These findings indicate that the hairless gene product plays a wider role in development than previously suspected. Dev Dyn 1999;216:113-126.

Genetic analysis of Gv1, a gene controlling transcription of endogenous murine polytropic proviruses.

Gv1 is a genetic locus that coordinately regulates the expression of multiple murine leukemia virus-related endogenous proviral sequences. A quantitative nuclease protection assay for typing Gv1 inheritance has been developed. Use of this assay demonstrates that Gv1 controls transcription of polytropic but not of modified polytropic endogenous proviruses. A combination of genetic techniques were used to map Gv1; these analyses demonstrate that Gv1 lies approximately 37 centimorgans from the centromeric end of mouse chromosome 13.

Host range and interference studies of three classes of pig endogenous retrovirus.

Recent interest in the use of porcine organs, tissues, and cells for xenotransplantation to humans has highlighted the need to characterize the properties of pig endogenous retroviruses (PERVs). Analysis of a variety of pig cells allowed us to isolate and identify three classes of infectious type C endogenous retrovirus (PERV-A, PERV-B, and PERV-C) which have distinct env genes but have highly homologous sequences in the rest of the genome. To study the properties of these env genes, expression plasmids for the three env genes were constructed and used to generate retrovirus vectors bearing corresponding Env proteins. Host range analyses by the vector transduction assay showed that PERV-A and PERV-B Envs have wider host ranges, including several human cell lines, compared with PERV-C Env, which infected only two pig cell lines and one human cell line. All PERVs could infect pig cells, indicating that the PERVs have a potential to replicate in pig transplants in immunosuppressed patients. Receptors for PERV-A and PERV-B were present on cells of some other species, including mink, rat, mouse, and dog, suggesting that such species may provide useful model systems to study infection and pathogenicity of PERV. In contrast, no vector transduction was observed on nonhuman primate cell lines, casting doubt on the utility of nonhuman primates as models for PERV zoonosis. Interference studies showed that the three PERV strains use receptors distinct from each other and from a number of other type C mammalian retroviruses.

Fv1, the mouse retrovirus resistance gene.

A number of genes which affect the susceptibility of mice to infection by retroviruses have been described. One of the most interesting of these genes is Fv1 (Friend virus susceptibility 1), which acts at a stage in the retroviral life-cycle following virus entry into the cell but prior to integration and formation of proviral structures. A detailed understanding of the mode of action of Fv1 might be expected to shed fresh light on early steps of the retroviral replication, although progress has been slow in this area due to uncertainty about the nature of the Fv1 gene. The recent cloning of Fv1 by a positional approach fills this gap in current knowledge. Fv1 appears to be derived from a fragment of a retroviral genome, an observation that may suggest novel approaches to the control of retroviral replication.

Endogenous retroviruses: a potential problem for xenotransplantation?

To overcome the shortage of suitable human donors for transplantation attention has recently turned to the possibility of using genetically modified pigs as a source of cells and organs. It has been suggested that such procedures might facilitate the introduction of novel retroviruses, normally resident in the pig germ line, into the human population (Stoye and Coffin, Nature Medicine 1: 1100, 1995). The consequences of such a transfer remain unclear; however, the demonstration that certain porcine cell lines express infectious retroviruses which can infect human cells (Patience et al., Nature Medicine 3: 282-286, 1997) emphasizes that there are grounds for practical concern. We have now cloned the envelope genes of the expressed viruses and are using these clones in studies of the interaction of the porcine viruses with their cellular receptors. We have also initiated studies of the inheritance and expression of human-tropic endogenous proviruses present in different pig populations. These studies reveal that at least two classes of human-tropic endogenous porcine retrovirus are widely distributed in pigs (Le Tissier et al., Nature 389: 681-681, 1997). The implications of our results for assessing the potential risk of retroviral transfer during xenotransplantation are discussed.

Endogenous retroviruses and the evolution of resistance to retroviral infection.

The current AIDS epidemic has rekindled interest in the evolution of retroviruses and the development of resistance to infection. Retroviruses and their vertebrate hosts have coexisted for millions of years, during which time a variety of host defence mechanisms has evolved. One repeated strategy is to use endogenous retroviruses to combat infection by their exogenous relatives.

Positional cloning of the mouse retrovirus restriction gene Fv1.

Vertebrate evolution has taken place against a background of constant retrovirus infection, and much of the mammalian genome consists of endogenous retrovirus-like elements. Several host genes have evolved to control retrovirus replication, including Friend-virus-susceptibility-1, Fv1, on mouse chromosome 4 (refs 3, 4). The Fv1 gene acts on murine leukaemia virus at a stage after entry into the target cell but before integration and formation of the provirus. Although restriction is not absolute, Fv1 prevents or delays spontaneous or experimentally induced viral tumours. In vitro, Fv1 restriction leads to an apparent 50-1,000 fold reduction in viral titre. Genetic evidence implicates a direct interaction between the Fv1 gene product and a component of the viral preintegration complex, the capsid protein CA (refs 7-9). We have now cloned Fv1: the gene appears to be derived from the gag region of an endogenous retrovirus unrelated to murine leukaemia virus, implying that the Fv1 protein and its target may share functional similarities despite the absence of nucleotide-sequence homology.

The genetics of tasting in mice. VII. Glycine revisited, and the chromosomal location of Sac and Soa.

Previous work which appeared to show that some strains of mice taste glycine solutions as bitter has been found to be in error. The bitterness came from copper glycinate which formed in the brass drinking spouts. Taste testing with copper glycinate shows that the genetical data identifying the gene Glb are still valid. The close linkage of Glb and Rua has been confirmed. Most strains of mice prefer glycine solution to water, presumably because the glycine tastes sweet. The degree of preference for glycine is correlated with the degree of preference for other sweet substances such as saccharin or acesulfame. The gene dpa appears not to be involved. The sweetness tasting gene Sac has been mapped to chromosome 4 at 8.1 +/- 3.4 cM distal to Nppa (formerly Pnd). The bitterness tasting gene Soa is very closely linked to Prp on chromosome 6 (no recombinants among 67 backcross progeny). It is suggested that the sweetness and bitterness tasting genes have descended from a common ancestral tasting gene which existed before the tetraploidization of the genome which took place in early vertebrate evolution.

Genetic map of the region surrounding the retrovirus restriction locus, Fv1, on mouse chromosome 4.

The Friend virus susceptibility-1 (Fv1) gene maps to mouse Chromosome (Chr) 4 close to a cluster of four endogenous murine leukemia viruses (MLVs). To investigate the feasibility of cloning Fv1 by a positional approach, we have performed an extensive genetic analysis of this region of Chr 4. We have typed 368 backcross mice for the four proviruses, Nppa, Lck, and D4Smh6b. Recombinant animals were screened in a hierarchical fashion with a variety of other markers, including Fv1 and the isozyme marker Gpd1. A detailed genetic map of the region surrounding Fv1 was derived. Three markers, Xmv9, Nppa, and Iap3rc11, were identified that showed no recombination with Fv1. By combining backcross and recombinant inbred strain data, we estimated that Xmv9 and Nppa must lie within 0.6 cM of one another and Fv1.