Feline immunodeficiency virus ORF-Ais 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 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.

FIG. 1.

FIV-pPPR orf-A mutants. All orf-A mutant proviruses were derived from pSV-pPPR, which is an FIV-pPPR-based provirus. The N-terminal acidic or hydrophobic, central leucine, and C-terminal cysteine regions, as well as conserved tryptophans, are indicated by solid rectangles. All deletions of Orf-A amino acids are indicated by dashes and are in frame. Dots represent amino acids identical to those present in WT Orf-A. Deletion of 13 residues (residues 24 to 36) and replacement of phenylalanine 23 by an aspartic acid (D) residue (introduced by the inserted ClaI restriction enzyme site) are shown for the ΔorfAmid mutant. All other amino acid substitutions (asterisks) are alanine substitutions.

FIG. 2.

Replication of pSV orf-A mutants in feline PBMC and MCH5-4 cells. CrFK cells were electroporated with pSV orf-A mutant provirus constructs or pSVWT and then cocultivated with either primary feline PBMC or MCH5-4 cells as described in the text. Cocultivated PBMC were separated from CrFK cells, maintained in culture up to 4 weeks posttransfection, and monitored by an FIV p24^gag antigen capture ELISA as described in Materials and Methods. Shown is the replication of all pSV orf-A-generated mutants in feline PBMC (A), of pSV orf-A mutants that are severely restricted in feline PBMC (B), of all pSV orf-A-generated mutants in feline MCH5-4 cells (C), and of pSV orf-A mutants that are severely restricted in MCH5-4 cells (D). Data are representative of three or more experiments.

FIG. 3.

Quantitation of intracellular FIV mRNA expressed by orf-A mutant proviruses. 3201-B cells were transfected with either an FIV-pPPR orf-A mutant provirus or WT FIV-pPPR. All proviruses were cotransfected with pNDeGFP to normalize transfection efficiencies. By 24 h posttransfection, total RNA was extracted from harvested cells and reverse transcribed to cDNA, and the copy number was determined by a real-time PCR assay as described in the text. The FIV RNA copy number per 200,000 GFP-positive cells was determined from cells transfected with FIV-pPPRΔorfAmid (A), FIV-pPPRorfA-L-A (B), FIV-PPRorfA-C-A (C), or FIV-pPPRorfA-WW/AA (D) and was compared to the FIV RNA copy number from cells transfected with WT FIV-PPR. Values shown are means of triplicate transfections except for the experiment showing FIV-pPPRΔorfAmid, which was performed in duplicate. Error bars, standard deviations. Data are representative of three experiments.

FIG. 4.

Orf-A mutation alters virus particle formation and is complemented by coexpression of GFP-Orf-A. 3201-B cells were transfected with either an FIV-pPPR orf-A mutant provirus or WT FIV-pPPR. All proviruses were cotransfected with pNDeGFP to normalize transfection efficiencies. Within 24 h posttransfection, total RNA was extracted from supernatants harvested from transfected cells, reverse transcribed to cDNA, and measured for FIV gag copy number by real-time RT-PCR as described in the text. Total RNA was also tested in the real-time PCR assay as a control for possible DNA contamination. The FIV RNA copy number per milliliter of cell culture supernatant from cells transfected with FIV-pPPRΔorfAmid (A), FIV-pPPRorfA-L-A (B), FIV-pPPRorfA-C-A (C), or FIV-pPPRorfA-WW/AA (D) was assayed and compared to the copy number per milliliter of supernatant from cells transfected with WT FIV-pPPR. Similarly, the FIV RNA copy number per milliliter of cell culture supernatant from cells transfected with either FIV-pPPRΔorfAmid or WT FIV-pPPR was assayed and compared to that measured from cells cotransfected with FIV-pPPRΔorfAmid and pEGFP-OrfA (E). 3201-B cells transfected with pNDeGFP alone served as a mock control. Data shown are representative of three or more experiments.

FIG. 5.

Infection of feline PBMC with orf-A mutant viruses. Feline PBMC (5 × 10⁶) were infected with WT FIV or FIV orf-A mutant inocula containing 2.5 ng of virion-associated FIV p24^gag. Mock infections served as negative controls. At 24 h postinfection, the inoculum was removed, genomic DNA was extracted from harvested cells, and the copy number was determined by a real-time PCR assay as described in the text. FIV DNA copies per 5 × 10⁶ cells infected with FIV-pPPRorfA-L-A (A), FIV-pPPRorfA-C-A (B), or FIV-pPPRorfA-WW/AA (C) inocula are shown. Genomic DNA samples were normalized by quantitation of cellular CCR5 DNA copy number. Values shown are means of triplicate infections. Error bars, standard deviations. Data shown are representative of three experiments.

FIG. 6.

Expression of FIV p24^gag by FIV-pPPR orf-A mutant proviruses. 3201-B cells were transfected with either WT FIV-pPPR (lane 1), FIV-pPPRΔorfAmid (lane 2), FIV-pPPRorfA-WW/AA (lane 3), FIV-pPPRorfA-C-A (lane 4), or FIV-pPPRorfA-L-A (lane 5). All proviral plasmids were cotransfected with pNDeGFP to normalize for transfection efficiency. Cells transfected with pNDeGFP alone served as a negative control. Within 48 h posttransfection, immunoprecipitations were performed on cellular extracts by using FIV-infected cat sera, and proteins were electrophoretically separated on SDS-12% polyacrylamide gels. Immunoblot analysis was performed by using a mouse monoclonal antibody against FIV p24^gag.