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91 Characterization of Functional Domains of the Feline Immunodeficiency Virus Accessory Gene orfA
M. Gemeniano*, J. Byerly, E. Sawai, and E. Sparger
Univ. of California, Davis, USA
Background: The feline immunodeficiency virus (FIV) is the etiological agent of a chronic, progressive disease found in cats that is similar to human AIDS caused by human immunodeficiency virus (HIV) infection. In addition to disease progression, FIV shares several genetic similarities with HIV and therefore provides an animal model for development of HIV vaccines and anti-viral therapies. The elucidation of viral determinants that are necessary for FIV viral replication, cell tropism, and pathogenesis is important for designing efficacious lentiviral vaccines and anti-viral drugs. Particularly important is the elucidation of function of genes necessary for robust viral replication. FIV orf-A is an accessory gene necessary for efficient virus replication both in vitro and in vivo. Although orf-A has been considered to be a putative tat gene for FIV, functions of orf-A remain unclear. The objective of this study is to delineate the main function of orf-A.
Methods: (1) To assess replication, provirus constructs with complete (pSV-?OrfA), partial (pSV-?OrfAmid), and point mutations in orf-A were constructed and gag expression assessed in feline PBLs and feline T-cell lines supernatant. (2) To assess transcriptional up-regulation, viral mRNA was measured from cell transfected with orf-A mutant provirus. (3) To assess viral infectivity, viral mRNA was measured from cells infected with orf-A mutant virions.
Results and Conclusions: OrfA mutants demonstrate a severely restricted replication in feline PBLs and feline T-cell lines. In addition, replication studies indicate that the central leucine-rich, C-terminal cysteine-rich, and the WW-like domains of orf-A, which are regions common to ungulate lentivirus Tat proteins, are critical for efficient replication of FIV. Interestingly, recent studies demonstrate a possible role of proteins containing WW domains in retroviral assembly and budding. OrfA mutants produced only 4- to 8-fold less viral transcripts than FIV wild type suggesting that the major function of OrfA may not be transcriptional. However, in experiments assessing infectivity of OrfA mutant virions, OrfA mutant virions produced 100-fold less transcripts than FIV wild type suggesting a post-transcriptional role for OrfA.
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