Development of Feline Immunodeficiency Virus ORF-A (tat) Mutants: In Vitro and in Vivo Characterization

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.

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Gemeniano¹,

Sawai

Leutenegger³

et al. 2003

“…The numbers of input copies were correctly estimated using the homologous oligonucleotide sets: FL-381 was underestimated by a factor of 10 6 with the GL8-specific oligonucleotides, and GL8 was underestimated by a factor of 10 4 with the FL-381-specific oligonucleotides (data not shown). Plasma samples were processed as described elsewhere (60). Briefly, RNA genomes were extracted from plasma by using the QIAamp Viral RNA kit (Qiagen, Milan, Italy), reverse transcribed with the antisense primer FL-381 AS (900 nM) or GL8 AS (300 nM), and then amplified with the sense primer FL-381 S or GL8 S (300 nM) and …”

Section: Methodsmentioning

confidence: 99%

“…Serial 10-fold dilutions (10 1 to 10 7 ) of FL-381 and GL8 gag p24 RNA transcripts were used to produce standard curves. Intra-and interassay precision and reproducibility were assessed as described previously (26,42,60). The sensitivities of both assays were 200 copies per ml of plasma, as evaluated by extracting and amplifying FIV-negative plasma spiked with serial 10-fold dilutions of RNA transcripts.…”

Section: Vol 77 2003 Protection By Fiv Vaccine From Intraclade Chalmentioning

confidence: 99%

AIDS Vaccination Studies Using an Ex Vivo Feline Immunodeficiency Virus Model: Protection from an Intraclade Challenge Administered Systemically or Mucosally by an Attenuated Vaccine

Pistello

Matteucci

Bonci

et al. 2003

Feline immunodeficiency virus (FIV) infection of domestic cats represents a valuable system through which to investigate criteria for antilentiviral vaccines in a natural host species. Here, we examined whether vaccination with a strain of FIV attenuated as a result of prolonged growth in vitro could protect against a fully virulent, highly heterologous intraclade challenge. The results indicated that the vaccine virus produced a low-grade infection with no detectable pathological effects and afforded a long-lasting sterilizing immunity if the challenge was delivered intraperitoneally as cell-free virus but not against a cell-associated intravaginal challenge. In the latter case, however, the replication and pathological consequences of the challenge virus were markedly suppressed. Together with similar results obtained in rhesus monkey models, these findings should give impulse to the development of attenuated FIV vaccines to be tested in controlled studies in field cats. Field studies may provide answers to some of the existing safety concerns surrounding attenuated AIDS vaccines in humans.

“…Pseudotyped FIV particles were prepared by cotransfecting 293T cells with vector, packaging, and Env plasmids. Packaging p⌬env1 was produced from p⌬00, a replication-competent molecular clone of FIV-Pet derived from p34TF10 (56). A detailed description of p⌬env1 was described elsewhere previously (55).…”

mentioning

confidence: 99%

Env-Expressing Autologous T Lymphocytes Induce Neutralizing Antibody and Afford Marked Protection against Feline Immunodeficiency Virus

Pistello

Bonci

Zabogli

et al. 2010

The envelope (Env) glycoproteins of HIV and other lentiviruses possess neutralization and other protective epitopes, yet all attempts to induce protective immunity using Env as the only immunogen have either failed or afforded minimal levels of protection. In a novel prime-boost approach, specific-pathogen-free cats were primed with a plasmid expressing Env of feline immunodeficiency virus (FIV) and feline granulocyte-macrophage colony-stimulating factor and then boosted with their own T lymphocytes transduced ex vivo to produce the same Env and interleukin 15 (3 ؋ 10 6 to 10 ؋ 10 6 viable cells/cat). After the boost, the vaccinees developed elevated immune responses, including virus-neutralizing antibodies (NA). Challenge with an ex vivo preparation of FIV readily infected all eight control cats (four mock vaccinated and four naïve) and produced a marked decline in the proportion of peripheral CD4 T cells. In contrast, five of seven vaccinees showed little or no traces of infection, and the remaining two had reduced viral loads and underwent no changes in proportions of CD4 T cells. Interestingly, the viral loads of the vaccinees were inversely correlated to the titers of NA. The findings support the concept that Env is a valuable immunogen but needs to be administered in a way that permits the expression of its full protective potential.