DE DERECHOS HUMANOS Y EN LA LEGISLACIÓN DE BRASIL Y MÉXICO
3.4. TRATADOS INTERNACIONALES
3.4.4 CONVENCIÓN AMERICANA DE 1969 (PACTO DE SAN JOSE)
3.4.4.1 CORTE INTERAMERICANA DE DERECHOS HUMANOS
Before introducing Gvl, it is important to consider the target for the gene, the endogenous MLVs, in more detail. The classification, mapping and characteristics of these elements will therefore be presented.
1.8.1 - Host range:
Endogenous MLV-related proviruses are classified according to the structures of their env
genes (Coffin, 1996). Viruses isolated from inbred strains of mice are separated into two main groups, ecotropic and non-ecotropic. The ecotropic class can only infect mouse (and rat) cells and can be transmitted vertically or horizontally. Consequently, both endogenous and exogenous examples have been described. The integration site of individual proviruses allow them to be identified based on the size of a particular junction fragment by Southern blotting techniques. In mice, this method was first used to identify and map specific ecotropic MLVs {Emv loci). These pro viruses are only present in low copy numbers, with
zero to ten copies present per haploid mouse genome (Jenkins et al. 1982). The first endogenous ecotropic retrovirus sequenced was AKV MLV, that was imphcated in the high incidence of thymic leukaemias in AKR mice (Herr 1984).
The non-ecotropic viruses are sub-divided into the xenotropic, poly tropic, modified polytropic and amphotropic groups. The xenotropic class can only replicate in non-mouse cells, due to incompatible receptors, which differ from those used by ecotropic viruses. Consequently, endogenous xenotropic viruses do not cause disease in inbred mice and can only be genetically transmitted. The second non-ecotropic group are typified by the MCF retroviruses (Hartley et al. 1977). These viruses are replication competent as a result of recombination between endogenous ecotropic and non-ecotropic viruses. They contain an altered env gene relative to ecotropic viruses which encodes the ability to infect both mouse and non-mouse cells (Khan 1984; Stoye et al. 1991a). The viruses are therefore known as dual tropic or more commonly, poly tropic. Structural analysis of a number of polytropic proviral sequences revealed that a third non-ecotropic class, the modified polytropic viruses, could be classified based on a characteristic env deletion (Stoye and Coffin 1987).
Amphotropic retroviruses are the final group of non-ecotropic viruses, which can also infect both mouse and non-mouse cells. No endogenous examples have been identified in inbred strains of mice. However, exogenous examples were isolated from California wild mice, a group of animals known to produce infectious amphotropic virus as a result of horizontal or congenital infection (O'Neill et al. 1987).
1.8.2 - LTR structure:
No infectious non-recombinant polytropic or modified polytropic proviruses have been isolated to date. This may reflect a combination of mutations and deletions in the coding sequences that render them replication incompetent, although it may also be caused by their LTR structures. Two studies simultaneously described the sequence of the U3-R regions of
proviral clones isolated from BALB/c mice that contained a 190 bp insert compared to the LTRs of xenotropic viruses (Khan and Martin 1983; Ou et al. 1983). It was shown that these longer LTR structures represented the endogenous polytropic and modified polytropic class of MLVs based on their corresponding env sequences (Stoye and Coffin 1987; Ch'ang
1989).
The U3 regions of both classes of provirus contain the TATA and CCAAT-like boxes found in ecotropic and xenotropic LTRs which are necessary for transcription (see section 1.7.1). However, one of the two 75 bp DRs found in Mo-MLVs is missing and a unique 14 bp DR is found towards the 5' end of U3 (see Figure 1.3). The 190 bp insert is bound by imperfect 6 bp repeats, which is a structure characteristic of transposable elements and alluded to the origin of this sequence (Khan and Martin 1983). Evidence was soon presented, however, that the insert resulted from recombination between the MuRRS group of elements and endogenous non-ecotropic LTRs (Schmidt et al. 1985). Two further deletions, of 5 and 39 bp, were observed in the U3 region of polytropic pro viruses compared to clones derived from the modified polytropic class (Stoye and Coffin 1987). The significance of these regions to this study will be discussed later.
1.8.3 - Origins:
Based on Southern blot analysis using type-specific probes from four different viral env
genes, it was shown that xenotropic and MCF viruses were more widely distributed in wild mice than the ecotropic class, suggesting that non-ecotropic viruses were integrated into the
Mus germline at an earlier date (Kozak and O'Neill 1987). This is consistent with the hypothesis that endogenous ecotropic viruses found in inbred strains of mice resulted from recombination between the env gene of non-ecotropic virus and another unknown virus that is not endogenous to the germline (Stoye and Coffin 1987). The sequence variation of the
env gene observed amongst ecotropic isolates also suggests that such a recombination event has occurred several times (Kozak and O'Neill 1987).
The evolutionary relationships of the endogenous MLVs were examined by comparison of the gp70 amino acid sequence. The members of the non-ecotropic class were closely related, as expected, although the ecotropic viruses showed only 58% sequence identity to the others. The evolutionary tree suggests that insertion of the 190 bp element into U3 presumably occurred sometime after the divergence of xenotropic and polytropic viruses, and that modified polytropic and polytropic viruses diverged before the generation of inbred strains (Stoye and Coffin 1987).
1.8.4 - Genetic mapping of endogenous MLVs:
Oligonucleotide hybridisation probes were designed in the env region to determine the relative distribution of the different classes of non-ecotropic endogenous proviruses in the commonly used inbred strains of mice. DNA from the relevant animals was digested with a restriction enzyme such as PvuII, and each provirus yielded a unique probe-reactive virus- host junction fragment, depending on the closest PvuII site in the flanking sequence (Stoye and Coffin 1988). Significant differences in proviral content between strains were observed using this technique, yet only a small proportion of individual proviruses were unique to a given strain. It was also estimated that between 40 and 80 copies of non-ecotropic proviral sequences were present per genome (Stoye and Coffin 1988). The relative structures of these four classes of endogenous MLV are shown in Figure 1.4. Using class-specific oligonucleotide probes, xenotropic pro viruses (Xmvs) were mapped by the segregation of junction fragments in recombinant inbred (RI) mouse strains (Frankel et al. 1989a), followed by the polytropic pro viruses (Pmvs) (Frankel et al. 1989b) and finally the modified polytropic pro viruses (Mpmvs) (Frankel et al. 1990). Over 160 individual loci have been now been mapped (Frankel et al. 1992). LTR probes have also been used more recently to map proviral loci. The fact that pro viruses contain duplicated LTRs meant that the hybridisation patterns were more complex than those with env probes. However, it could be concluded that less than 10% of the MLV sequences present in the genome are represented by solo LTRs (Frankel and Coffin 1994).
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X CÛ .52, LU - CÛ CÛ CÛ CÛ OÛ in LU CÛ X OÛ CÛ (/) > c 0) o CL O) 0 5 O)Figure 1.4: S tructures of endogenous MLVs. Features indicated are the restriction sites for EcoRI (E), BamHI (B), and Hindlll (H). The relative sizes of the LTRs with the 150 bp and 190 bp inserts in poly tropic and modified polytropic pro viruses (shaded) are shown; the positions of the probes specific for ecotropic (pEco), xenotropic (js6/10), polytropic (js5) and modified polytropic (js4) sequences are indicated. Redrawn from Stoye and Coffin (1988).
From these studies it was concluded that members of individual strains contain an identical complement of endogenous proviruses and that few were unique to one particular strain, implying that modem inbred strains of mice are derived from a small pool of animals. These proviruses also appear to be evenly distributed across the mouse genome, apart from four xenotropic loci tightly linked to the retroviral restriction locus, Fvl (Stoye et al. 1995).
1.8.5 - Acquisition and loss of endogenous MLVs:
Few examples of proviral movement have been documented, suggesting that these elements are stable in the mouse genome. Germhne movement of proviruses has been most extensively studied in the 17 substrains of the AKR mouse that have been identified. Of the few ecotropic proviruses expressed, only one pro virus (E m vll) was found in all the strains analysed, suggesting that E m vll existed in the original AKR stock. (Buckler et al. 1982). It has since been calculated that AKR mice gain one ecotropic pro virus for every 50 to 100 generations. Even higher frequencies of acquisition have been observed in SWR/J-RF/J hybrid mice, where as many as 10 ecotropic proviral integration sites were observed in a single animal from some Utters and three-quarters of the progeny obtained new proviruses (Jenkins and Copeland 1985). Acquisition of non-ecotropic pro viruses appears to be occurring approximately 50-times more slowly, based on the data from all the RI strains analysed to date (Frankel et al. 1990).
Analysis of ecotropic proviral acquisition in mice demonstrated that reverse transcription and infection stages are required for the addition of novel pro viruses. Ovaries from mice with no endogenous ecotropic pro viruses were transplanted into female hosts that carried two Emvs.
The mice were crossed with virus-negative males, and novel proviruses were detected in progeny derived from the donor (virus negative) ovary. These proviruses were therefore derived from infection, since none could possibly have been inherited (Lock et al. 1988). In addition, intracellular retrotransposition may account for the insertion of new pro viruses.
which has been shown by in vitro studies using constructs derived from lAPs (Heidmann and Heidmann 1991).
The loss of proviral sequences has also been observed, although all of the examples documented to date resulted from homologous recombination between two LTRs. In the case of the d mutant in mice, loss of an ecotropic provirus causes reversion of the mutant phenotype, leaving behind a characteristic solo LTR (Copeland et al. 1983). An estimate of the excision rate was made based on the number of d mutants observed in the inbred mouse population, of one event per 5 million meiosis (Seperack et al. 1995).
In summary, the organisation, distribution and movement of endogenous MLVs have been studied in detail over the last 20 years. Their structure is relatively homogeneous, and their apparent stability in the genome means they are suitable molecular markers for evolutionary studies of the mouse. The challenge is now to understand more about the mechanisms of endogenous proviral expression and how this may relate to disease in both mouse and human.