Recombination between feline leukemia virus subgroup B or C and endogenous env elements alters the in vitro biological activities of the viruses

Abstract

An important question in feline leukemia virus (FeLV) pathogenesis is whether, as in murine leukemia virus infection, homologous recombination between the infecting FeLV and the noninfectious endogenous FeLV-like proviruses serves as a significant base for the generation of proximal pathogens. To begin an analysis of this issue, several recombinant FeLVs were produced by using two different approaches: (i) the regions of the viral envelope (env) gene of a cloned FeLV (subgroup B virus [FeLV-B], Gardner-Arnstein strain) and those of two different endogenous proviral loci were exchanged to create specific FeLV chimeras, and (ii) vectors containing endogenous env and molecularly cloned infectious FeLV-C (Sarma strain) DNA sequences were coexpressed by transfection in nonfeline cells to facilitate recombination. The results of these combined approaches showed that up to three-fourths of the envelope glycoprotein (gp7O), beginning from the N-terminal end, could be replaced by endogenous FeLV sequences to produce biologically active chimeric FeLVs. The in vitro replication efficiency or cell tropism of the recombinants appeared to be influenced by the amount of gp7O sequences replaced by the endogenous partner as well as by the locus of origin of the endogenous sequences. Additionally, a characteristic biological effect, aggregation of feline T-lymphoma cells (3201B cell line), was found to be specifically induced by replicating FeLV-C or FeLV-C-based recombinants. Multiple crossover sites in the gp7O protein selected under the conditions used for coexpression were identified. The results of induced coexpression were also supported by rapid generation of FeLV recombinants when FeLV-C was used to infect the feline 3201B cell line that constitutively expresses high levels of endogenous FeLV-specific mRNAs. Furthermore, a large, highly conserved open reading frame in the pol gene of an endogenous FeLV provirus was identified. This observation, particularly in reference to our earlier finding of extensive mutations in the gag gene, reveals a target area for potentiafly productive homologous recombination upstream of the functional endogenous env gene. Multiple steps are involved in leukemogenesis caused by infection of the host by replication-competent retroviruses. Among the mammalian retroviruses, considerable information on murine leukemia virus (MuLV)-induced leukemia, which appears to produce several characteristic virus-host interactions, has been generated. The envelope protein gene (env) of the infectious MuLV recombines with the endoge-nous MuLV-related sequences inherited in the mouse ge-nome and expressed in mouse cells to give rise to recombi-nant MuLVs that are capable of recognizing different sets of cell surface receptors. Such altered recognition may be critical in establishing infection in various host cells including the target cells (11, 14, 28, 37). There is also evidence for insertional mutagenesis that frequently activates proto-on-cogenes like c-myc, pim-J, pvt-J, etc., in tumor cells (4). Additionally, hematopoietic hyperplasia, cytopathic effect on bone marrow cells, and other related effects induced directly or indirectly by MuLV infection appear to contribute to the development of leukemia (9, 20). While these observed phenomena offer opportunities for a critical analysis of the mechanism of the disease in the mouse system, there is only limited information on feline leukemia virus * Corresponding author. t Present address: (FeLV) infection, which naturally and frequently results in leukemogenesis in the domestic-cat population. Analogous to the mouse model, FeLV infection predominantly causes T-cell lymphoma in cats after a prolonged latency (13, 16, 21), and some recent studies point to potential interactions between exogenous and endogenous FeLV elements (19, 25). Furthermore, evidence that indicates frequent activation of c-myc in feline T lymphomas by either viral trans-duction or insertional mutagenesis (22) has accumulated. To understand the nature of interaction between the infecting strain of FeLV and transcriptionally active but replication-defective (5, 23, 24, 35, 36) endogenous FeLV elements of the domestic-cat genome, which may be responsible for the generation of proximal leukemogens, it is necessary to delineate the effects contributed by specific domains of the endogenous sequences. With this aim, we generated chimeric FeLVs to examine their biological activities. In vitro infectivity studies with individual or mixtures of recombinants show that up to three-fourths of the envelope surface glycoprotein (gp7O), beginning from the N-terminal end, could be replaced by endogenous FeLV sequences to produce biologically active chimeric FeLVs, although the efficiency of infection of these chimeras in certain cell types appears to be influenced by the locus of origin of the endogenous component as well as by the length of the endogenous insert. Multiple crossover sites on the env gene selected in the recombination process have also been identified. These findings along with that of a large open 6495