Potential Protein Coding Regions in FIV Negative Strand RNA
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DESCRIPTION (provided by applicant): Retroviruses have relatively simple plus-strand RNA genomes that have the coding capacity for the essential proteins that make up the virus particle, including the structural proteins (encoded by gag); the enzymes required for replication and assembly (encoded by pol); and the receptor binding protein(s) that facilitate entry of the target cell (encoded by env). With the characterization of the lentiviruses, it was recognized that other ancillary proteins that provide selective advantage for the virus in particular cell environments and in the face of host defenses are also encoded via spliced messages. These (known) proteins include Vif, Tat, Rev, and in the non-primate lentiviruses including FIV, deoxyuridine triphosphatase (DU), all encoded by the plus strand RNA transcript of the lentivirus genome. In the present proposal, we wish to pursue earlier studies that suggested that FIV may encode a protein or proteins from an anti-sense message, an idea inspired by recent observations by others that HIV may encode an antisense protein (ASP) and that this protein may influence autophagy. To this end, we propose to (1) perform alignments of the negative strands of known FIV genomes and identify potential regions that might be translated into protein; (2) clone and express candidate peptides/proteins revealed in (1) for antibody preparation in order to test for the presence of such proteins/peptides in FIV-infected target cells; (3) transiently express these putative proteins in FIV target cells and assess influence on cell viability/function and on virus expression; and (4) prepare mutant FIVs where ASPs are disrupted by stop codon(s) without interrupting the corresponding plus strand translations and assess influence of these mutations on virus expression. These studies should answer the question as to whether ASPs are produced in FIV infection and if so, suggest potential relevance to virus expression and host cell responses.
