Feline Immunodeficiency Virus Orf-A Is Required for Virus Particle Formation and Virus Infectivity

Abstract

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 FIVpPPR-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, virionassociated 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.

AIDS Vaccination Studies with an Ex Vivo Feline Immunodeficiency Virus Model: Analysis of the Accessory ORF-A Protein and DNA as Protective Immunogens

“…Initially considered the equivalent of the HIV-1 Tat, ORF-A has since been shown to (16); thus, no HIV-1 homologue to this protein can be currently identified. It is, however, well documented that an intact ORF-A is essential for FIV replication in CD4 ϩ T cells, a major target of in vivo infection, and an important determinant of FIV virulence, as shown by the markedly attenuated phenotype of viral mutants with the ORF-A gene deleted (15,36,38). Thus, we speculated that an immune response elicited against this protein might contain viral replication in CD4 ϩ T cells, hence sparing these cells from depletion and reducing the overall impact of infection on immune functions.…”

“…In particular, the accessory protein ORF-A, investigated as an immunogen in this report, is a small highly basic hydrophobic protein in which a few functional domains can be recognized. The central leucine-rich and Cterminal cysteine-rich domains are essential for viral replication in feline PBMC and T-cell lines (15), and the basic domain is important for cell localization (16). Initially considered the equivalent of the HIV-1 Tat, ORF-A has since been shown to (16); thus, no HIV-1 homologue to this protein can be currently identified.…”

“…Importantly, deletion of the ORF-A gene results in the production of viruses impaired for replication in lymphocytes and with reduced virulence. These characteristics provide ORF-A-deleted constructs with appropriate prerequisites for vaccine candidates (15,36,38).…”

Vaccination of Cats with Attenuated Feline Immunodeficiency Virus Proviral DNA Vaccine Expressing Gamma Interferon

“…Genomic DNA was isolated from PBMC using a QIAamp DNA blood mini kit (QIAGEN). Plasma RNA and PBMC DNA samples were assayed for FIV nucleic acid copy numbers using reverse transcription (RT) and quantitative real-time TaqMan PCR assays conducted on a 7700 ABI Prism sequence detector (Applied Biosystems, Foster City, CA) with FIV gag TaqMan probes and primer sequences by protocols previously described (16,31).…”

Accessory Genes Confer a High Replication Rate to Virulent Feline Immunodeficiency Virus

“…However, the FIV genome does include a multifunctional 77-amino-acid nonstructural viral protein expressed from the orfA gene, located between vif and env. OrfA is necessary for efficient FIV replication in lymphocytes in vitro and in vivo (52,(73)(74)(75)(76). Multiple potential functions have been ascribed to OrfA, including transactivation of viral protein expression (77), effects on virion formation and infectivity (73), cell cycle arrest (78), effects on cellular gene expression (79), and downregulation of cell surface expression of the primary FIV receptor, CD134 (80).…”