Abstract
Abstract
Recent studies on the molecular properties of feline immunodeficiency virus (FIV) and the natural history of the infection in cats represent a good model to understand the pathogenetic mechanisms induced by human immunodeficiency virus (HIV) and to develop new therapeutic and vaccinal approaches in the treatment of immunodeficiency syndromes. Recently, among the different vaccinal strategies tested, attenuated vaccines consisting of viruses deleted in one or more viral genes not essential for replication but important for pathogenicity have been shown to be really promising. In FIV, the accessory gene ORF-A is essential for wild type virus replication in lymphocytes. On the contrary, ORF-A-deleted viruses retain their replication capability only in macrophages. Molecular clones of FIV deleted in ORF-A and inoculated in vivo showed reduced pathogenicity and, when tested as vaccines, they provided partial protection against challenge with a virulent FIV strain. These ORF-A mutated molecular clones have been subjected to further genetic modifications to increase their immunogenicity and improve their vaccinal properties.
Aim of this study is the development of molecular clones of FIV deleted in ORF-A and containing mutations in the env gene coding for the viral surface glycoproteins. During the viral replication cycle lentiviral Env proteins are exposed on the surface of the infected cell from where, through endocytosis, they are partly addressed to cytoplasmic vesicles and degraded. We therefore hypothesized that a block in the endocytosis process should allow an increased expression of Env on the surface of the infected cells and of mature virus particles, thus increasing the immunogenicity of ORF-A mutants. Endocytosis occurs through a signal-sequence localised in the intracytoplasmic portion of Env, conserved among all lentiviruses and containing a tyrosine-glycine amino acid motif. To test this hypothesis we used two molecular clones:
III
Abstract
∆00, containing the functional ORF-A gene and ∆23 expressing a truncated ORF-A protein. Both clones ∆00 and ∆23 have been mutated in the signal-sequence for the endocytosis of Env; one mutation consists of the deletion of the codons for glycine and tyrosine amino acids designated (XX); the other consists of their substitution respectively with alanine and isoleucine designated (AI). We thus obtained four replication-competent molecular clones: ∆00XX, ∆00AI, ∆23XX and
∆23AI. All mutant clones were monitored in parallel with their parental viruses with unmodified env for kinetics of the endocytic process and analysed for surface expression of Env in infected cells compared to the control clones.
IV
