All mammalian species contain a very large number of endogenous retrovirus elements acquired by germline infection over the course of evolution. In humans retroviral elements represent approximately 8% of the genome and their number approaches the total number of encoded genes. Many endogenous retrovirus elements are defective, however some contain an entire complement of functional viral genes characteristic of replication-competent exogenous retroviruses. Although the expression of most endogenous retroviruses is largely suppressed in the host, the production of retroviruses or their gene products during development and during certain physiological or pathological states is well documented. Little is known about the control of retrovirus expression or the influence of such expression on the physiology or pathology of the host. An extensively investigated group of endogenous retroviruses are those giving rise to polytropic murine leukemia viruses (MuLVs) in mice. Polytropic MuLVs are formed by recombination of exogenous ecotropic MuLVs with endogenous envelope sequences present in the genomes of inbred mouse strains. The recombinant polytropic viruses exhibit an altered infectious host range and utilize a cell surface receptor distinct from the receptor utilized by ecotropic MuLVs. In several instances polytropic MuLVs have been directly implicated in pathogenesis, including the induction of proliferative, immunological, and neurological disorders. The generation of variants after infection results in a mixed retrovirus infection. One aspect of this project involves studies of the interactions of retroviruses in mixed infections. Upon co-inoculation of polytropic and ecotropic MuLVs in mice we have observed profound effects on the infectious spread of the polytropic virus, concomitant with a greatly enhanced neuropathogenicity. This phenomenon appears to be mediated by packaging of polytropic viral genomes within ecotropic virus particles. Another aspect of our studies involves the characterization of the family of endogenous retroviruses that exhibit gene sequences that are homologous to the sequences found in recombinant polytropic MuLVs and their participation in recombination. The participation of these endogenous retroviruses in recombination is not random and is different upon infection of mice with different exogenous viruses. We have characterized the endogenous retroviruses in NFS/N mice that could potentially give rise to the recombinant viruses and found that each could be distinguished by small nucleotide sequence differences. Comparison of the endogenous viruses to recombinant viruses derived after inoculation of an exogenous ecotropic MuLV indicated that only 3 of the approximately 25 to 30 endogenous retroviruses frequently participate in recombination. Furthermore, these studies suggested a likely mechanism for the specificity of recombination observed with different exogenous viruses. In additional studies we have found upon infection of mice with exogenous viruses, that viral sequences characteristic of intact mouse endogenous retroviruses are transferred to and replicate in cell lines derived from other species. The mobilization of endogenous retroviruses by infection with exogenous retroviruses is unprecedented and may have important implications for the involvement of endogenous retroviruses in disease processes. FY 2003 advance:All mammalian species contain a large number of retrovirus elements in their genomes that have accumulated by germ line infection over the course of evolution. The elements are expressed in a controlled manner during development and in certain pathological conditions and may play physiological or pathological roles in the host. We have characterized a group of endogenous retrovirus sequences in NFS/N mice that are known to be participants in the induction of proliferative diseases as well as other disorders. Their particcipation is mediated by recombination with exogenous retroviruses to generate new viruses, termed polytropic MuLVs that exhibit an altered host range of infection. It is known that the endogenous polytropic proviruses are not equally active in recombination and that recombination differs with different exogenous viruses. It is not known, however, precisely which of the endogenous elements recombine or are expressed in the host. This has been elusive because of the very close similarity of the endogenous sequences to one another. Our findings indicate that the proviruses are distinguishable by sequence heterogeneity. In addition, three distinct groups of proviruses were described, one of which is ancestral to the other groups. The latter group reflects features of a phylogenetic intermediate linking the polytropic MLVs to another MLV group termed the xenotropic MLVs. This study has identified distinguishing characteristics of the proviruses that should facilitate a more precise description of their expression in mice and their participation in recombination to generate new retroviruses, and also provided insight into the evolution of endogenous retroviral elements in the host. Methods: Tissue culture, retrovirus infectious assays,PCR,RT PCR,DNA sequencing, electrophoresis, immunohistochemistry. Goals and Objectives: A major goal of this project is the elucidation of the effects of genetic alterations of retroviruses on infection and the induction of pathology. Towards this end we hope to better understand the generation of retrovirus variants by point mutation and recombination. Other goals of this project are to gain a better understanding of the origins of endogenous retroviruses and to determine characteristics that facilitate their expression in the animal as well as their participation in recombination to generate variants. Research Significance: Retrovirus infection and replication in the host leads to genetic variation. Such variation can be the result of point mutations during replication, or recombination with endogenous retrovirus genes that are present in all mammalian species. A consequence of variation in the host is a mixed retrovirus infection. Examples of mixed virus infections resulting from genetic alteration include the generation of HIV variants in humans as a result of point mutation and the generation of polytropic viruses in mice by recombination of inoculated ecotropic murine retroviruses with endogenous retroviral genes. Although interactions of retroviruses are well documented, a role for such interactions in the replication and pathogenicity of the viruses is not well understood. Our studies provide a clear demonstration that mixed retrovirus infections can profoundly influence the replication and pathogenicity of retroviruses. Our studies of the endogenous retroviruses in mice provided important insights into their evolutionary relationships and facilitated a more complete description of the recombination process in the host. The finding that intact endogenous retroviruses may be mobilized by infection with exogenous viruses may have far reaching implications regarding the role of endogenous retroviruses in pathogenic processes. Future Plans: The finding that most endogenous polytropic retroviruses in mice are distinguishable by sequence heterogeneity enabled us to precisely identify the three endogenous retroviruses that frequently participate in recombination and to gain insight into structural features that influence the specific interaction of different endogenous retroviruses with different exogenous viruses. Why only 3 endogenous polytropic retroviruses among the 25 to 30 present in the genome participate in recombination is not yet known. We intend to precisely identify the endogenous retroviruses that are expressed in uninfected mice as well as mice infected with exogenous retroviruses. These analyses will determine if the participation of the endogenous retroviruses in recombination reflects their expression in the mice and if this expression is altered by exogenous virus infection. The observation that intact endogenous retroviruses may be mobilized by exogenous retrovirus infection is unprecedented and may have implications in the involvement of endogenous retroviruses in disease processes. Additional studies into this phenomenon will identify which endogenous retroviruses are mobilized by exogenous retrovirus infection and if the endogenous viruses spread in the host.
