Thesis (Ph.D.)--University of California, Davis, 2011
Feline immunodeficiency virus (FIV) is a member of the lentivirus subfamily of retroviruses and naturally infects domestic cats resulting in an immunodeficiency similar to that induced by human immunodeficiency virus (HIV) in humans. Previous research examining an accessory protein (Orf-A) encoded by FIV revealed a critical importance for this viral protein in virus replication under natural infection. Additional studies characterizing specific functions of Orf-A established that this viral protein shares similar functional characteristics as previously described for HIV-1 Vpr. To explain the disparity between the number of regulatory and accessory proteins encoded by FIV (3) in comparison to HIV-1 (6), we investigated Orf-A as a functional analog to HIV-1 accessory proteins Vpr as well as other HIV-1 accessory proteins
Previous studies identified a putative nuclear localization signal (NLS) and demonstrated Orf-A as a nuclear protein. To further assess the importance of residues within the putative NLS, amino acid substitutions were introduced as point mutations within the Orf-A NLS to examine their impact on nuclear localization. Results from these studies confirmed that basic residues within the putative NLS are necessary for nuclear localization. Examination of a FIV Orf-A NLS mutant provirus revealed that nuclear localization is critical function for efficient productive virus replication. Additional studies were conducted to further examine the role of Orf-A in the induction of arrest at gap 2 (G2) phase of the cell cycle and a possible role in apoptosis. Orf-A deletion mutant analysis revealed that amino residues 1-54 are critical for induction of G2 cell cycle arrest and induction of apoptosis comparable in magnitude to wild type (WT) Orf-A. Preliminary studies assessing a potential mechanism for Orf-A induction of apoptosis revealed a possible co-localization of Orf-A with the mitochondria compartment of host cells suggesting that further examination of this phenomenon is warranted. Additional assays of a feline lymphoid cell line experimentally infected with FIV revealed that FIV infection as well as transient Orf-A expression was capable of inducing G2 arrest. Furthermore, down-regulation of FIV primary receptor CD134 was also demonstrated for experimental FIV infection. Lastly, down-regulation of CD134, a recently described function of Orf-A, was confirmed by transient expression of Orf-A in a feline lymphoid cell line and shown to require the N-terminus of Orf-A of Orf-A. Mapping of Orf-A determinants for previously described Orf-A functions and discovery of new functions supports Orf-A as a multi-faceted accessory protein encoding functions described for HIV-1 accessory proteins including Vpr, Vpu, and Nef
