RESUMO
Feline immunodeficiency virus (FIV) gene orf-A, also designated orf-2, encodes a 77 amino acid accessory protein reported to be critical for efficient viral replication in vitro and in vivo and previously implicated to encode a Tat protein for FIV. However, recent studies have shown Orf-A to be important in the late steps of the FIV life cycle involved in virion formation and in early steps involved in virus infectivity. The present study reports that expression of a GFP-Orf-A fusion protein in both primate and feline cell lines results in nuclear localization of this FIV accessory protein. Moreover, a nuclear localization signal (NLS) critical for nuclear import was mapped to amino acid residues 43 through 53 of Orf-A. Lastly, transient expression of GFP-Orf-A in cells induced an arrest at the second gap (G(2)) of the cell cycle. Our findings reveal that Orf-A is a nuclear protein that expresses properties similar to those reported for human immunodeficiency virus-1 (HIV-1)-encoded Vpr.
Assuntos
Vírus da Imunodeficiência Felina/fisiologia , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Ciclo Celular , Núcleo Celular/virologia , Genes Virais , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/patogenicidade , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear/genética , Fases de Leitura Aberta , Mapeamento de Peptídeos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/genéticaRESUMO
The orf-A (orf-2) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within orf-A were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within orf-A severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in orf-A imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with orf-A mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from orf-A mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene vpr, vpu, or nef than to the regulatory gene tat encoded by the primate lentiviruses.