J P Stoye

Highly preferred targets for retrovirus integration

A central feature of retrovirus replication is integration of the provirus into host cell DNA, but the specificity of this step for cell target sequences has not been clarified. To investigate this issue, we developed a method for screening and comparing large numbers of unselected integration events. Using a replication-competent Rous sarcoma virus containing a bacterial suppressor tRNA gene as a selectable marker, we obtained collections of clones comprising integrated provirus together with host flanking sequences. Hybridization and sequence analysis of the flanking sequence reveals the presence of a number of strongly preferred integration targets. Within these targets, independent integration events occur at sites identical to the base.

Role of endogenous retroviruses as mutagens: The hairless mutation of mice

We have developed an experimental approach to distinguish the 40-60 endogenous C-type proviruses of mice and to determine their association with well characterized developmental and physiological mutations. The hairless (hr) mutation causes a variety of pleiotropic effects. Using oligonucleotide probes specific for different classes of murine leukemia virus, we have identified and cloned a provirus present in HRS/J hr/hr animals but absent in HRS/J +/+. Genetic analyses showed perfect concordance between the hr phenotype and the presence of the provirus in a number of inbred and congenic strains of mice. Molecular analysis of a haired revertant established the causal relationship since it revealed the excision of most of the proviral genome leaving behind one long terminal repeat. These findings show that virus integration caused the hairless mutation and point to the utility of naturally occurring retroviral integrations for accessing the genome of the mouse.

Mouse chromosome 4.

This year’s report incorporates 78 new genetic markers into the consensus linkage map. Of these markers, ten have a known, mapped human homolog. The murine gene, followed by the human homolog and the human chromosomal location in parenthesis are as follows: Cpt2 4 CPT2 (1p32); Cyp2j5 and Cyp2j6 4 CYP2J2 (1p31.3-p31.2); Guca1b 4 GUCA2B (1p34-p33); Htr6 4 HTR6 (1p36-1p35); Hub 4 ELAVL2 (9p21); Hud 4 ELAVL4 (1p34); Matn1 4 MATN1 (1p35); Mmp16 4 MMP16 (8q21.3-q22.1); Tgfbr1 4 TGFBR1 (9q33-q34). In the case of the Cyp genes, two genes have been identified in mouse while only a single gene has been identified in humans. Mouse Chromosome (Chr) 4 shares significant stretches of linkage homology with human Chr 1, 6, 8, 9 and 21 (34245; 23572). The entire distal half of mouse Chr 4 is homologous with human Chr 1p. There have been four changes in nomenclature of genetic loci on Chr 4. Gene names and/or symbols that have been changed by the Nomenclature Committee include: Cerr1 changed to Cer1; Dana changed to D4H1s1733E; Zie changed to Gklf; and Etl2 changed to Il11ra2.

A provirus put to work.

Retroviruses incorporate themselves into a host genome in the form of a provirus, and can be passed on to the hosts offspring. Evidence now emerges that one such provirus has been subverted to benefit of humans and other mammals that have a placenta — its protein product, called syncitin, may play an essential part in placental formation.

Characterization of the endogenous nonecotropic murine leukemia viruses of NZB/BINJ and SM/J inbred strains

We characterized 84 endogenous nonecotropic proviruses of NZB/B1NJ and SM/J inbred strains by examining proviral junction fragment segregation in recombinant inbred (RI) and backcross mice. Forty-five proviruses were shared with other laboratory strains, but 28 were unique to NZB/BINJ or SM/J. Proviral loci were located on 17 of the 19 mouse autosomes and on both sex chromosomes. These markers will facilitate gene mapping in the NXSM RI set and contribute to the pursuit of a more complete map of the mouse genome.

Genetics of Endogenous Murine Leukemia Viruses

Inbred strains of mice contain in the genome 40-60 endogenous proviruses related to murine leukemia virus. To assess the genetic and pathogenic consequences of these to the host, we have developed a strategy to distinguish among the three different host-range subgroups--xenotropic, polytropic and modified polytropic--by using oligonucleotide probes specific for a polymorphic region in env. Each of these proteins detects a relatively small number of bands in a Southern blot, thus permitting us to enumerate all individual proviruses of this group. Using this approach, we have determined the distribution of different proviruses among inbred and recombinant inbred (RI) strains congenic or coisogenic for specific mutants. Using the RI results, we have been able to place over 100 proviruses on the mouse genetic map. A number of these are closely linked to well-characterized mutations, and we have been able to establish that at least one mutation, hr (hairless), was caused by a proviral insertion. If the other close linkages also prove to reflect causality, we estimate that up to 5% of recessive mutations in the mouse might be caused by insertion of proviruses of this group. Using a similar probe strategy, we have followed the evolution of murine leukemia viruses during spontaneous leukemogenesis in AKR mice. We have found that the final leukemogenic (MCF) virus is a recombinant of three different endogenous parents; an ecotropic virus, a polytropic virus that directs the gp70 region of env, and a xenotropic virus (identified as the inducible element Bxv-1) that directs the LTR. In addition to the recombinations, all such viruses also have a reduplication of the enhancer region of the LTR, compared to the endogenous parent. MCF viruses are created by these three genetic changes, which occur in a reproducible fashion and appear in the thymus between 10 and 14 weeks of age.