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Journal of Virology, August 2003, p. 8819-8830, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.8819-8830.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Malou C. Gemeniano,1 Earl T. Sawai,2 Christian M. Leutenegger,1 and Ellen E. Sparger1*

Department of Medicine and Epidemiology, School of Veterinary Medicine,1 Department of Medical Pathology, School of Medicine, University of California, Davis, California 956162

Received 30 January 2003/ Accepted 15 May 2003

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.

* Corresponding author. Mailing address: 2108 Tupper Hall, Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616. Phone: (530) 754-8461. Fax: (530) 752-0414. E-mail: eesparger{at}ucdavis.edu.

Journal of Virology, August 2003, p. 8819-8830, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.8819-8830.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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