The nucleotide sequence and spliced pol mRNA levels of the nonprimate spumavirus bovine foamy virus

Abstract

We have determined the complete nucleotide sequence of a replication-competent clone of bovine foamy virus (BFV) and have quantitated the amount of splice pol mRNA processed early in infection. The 544-amino-acid Gag protein precursor has little sequence similarity with its primate foamy virus homologs, but the putative nucleocapsid (NC) protein, like the primate NCs, contains the three glycine-arginine-rich regions that are postulated to bind genomic RNA during virion assembly. The BFV gag and pol open reading frames overlap, with pro and pol in the same translational frame. As with the human foamy virus (HFV) and feline foamy virus, we have detected a spliced pol mRNA by PCR. Quantitatively, this mRNA approximates the level of full-length genomic RNA early in infection. The integrase (IN) domain of reverse transcriptase does not contain the canonical HH-CC zinc finger motif present in all characterized retroviral INs, but it does contain a nearby histidine residue that could conceivably participate as a member of the zinc finger. The env gene encodes a protein that is over 40% identical in sequence to the HFV Env. By comparison, the Gag precursor of BFV is predicted to be only 28% identical to the HFV protein.

FIG. 1

Genomic organization of BFV. The open reading frames for BFV were determined from the DNA sequence of λ BSV-11 described by Renshaw et al. (38). The approximate positions of the viral splice donor (SD), splice acceptor site for pol mRNA (SA), the Gly-Arg-rich region in Gag (GR), and the internal promoter (IP) are shown.

FIG. 2

Alignment of glycine-arginine-rich regions (GR boxes 1 to 3) within the putative NC proteins of spumaviruses (38). Amino acid residues that are conserved in the foamy virus NC proteins are in boldface and underlined. The amino acid positions of the GR boxes within the respective Gag polyproteins flank the amino acid sequences.

FIG. 3

Alignment of the pol mRNA splice junctions of BFV, HFV, and feline foamy virus (FeFV). Conserved nucleotides are shown in boldface. The nucleotide positions of the BFV splice donor and acceptor are shown above the sequence.

FIG. 4

Alignment of foamy virus Env proteins. Identical amino acids conserved among the foamy viruses are shown in bold. Putative functional domains are shown; the putative leader peptide is underlined with dashes, the proposed SU-TM cleavage site is marked by asterisks, the cell fusion domain is marked by number signs, the transmembrane segment is marked by plus signs, and the basic cytoplasmic tail is marked by carets. The amino acid positions of the Env proteins are to the right of the sequences.

FIG. 5

Alignment of foamy virus internal promoters. The nucleotide position of the first base from the start of transcription is shown on the right. Conserved nucleotides are in boldface. The TATA box and the starts of HFV (⇑) and SFV-1 (↑) transcription are underlined, as is the putative transcription start in BFV.

FIG. 6

Competitive PCR determination of the levels of gag, spliced pol, and Borf-2 specific mRNAs from cells collected after 4 days of cocultivation. The amount of specific competitor DNA added to each PCR is shown above appropriate gel lanes. Lanes C show the no-DNA-added PCR control. Five microliters of each PCR was cut in 20 μl with either BamHI (B) or BglII (G) and run on a 3% agarose gel for analysis. (A) Spliced pol mRNA; (B) gag mRNA; (C) Borf-2 mRNA.

FIG. 7

Phylogenetic tree. Amino acid sequences representing segments of IN (44) were analyzed with software from DNAStar (41).