Foamy virus integration

Abstract

It had been suggested that during integration of spumaretroviruses (foamy viruses) the right (U5) end of the cDNA is processed, while the left (U3) remains uncleaved. We confirmed this hypothesis by sequencing two-long terminal repeat (LTR) circle junctions of unintegrated DNA. Based on an infectious foamy virus molecular clone, a set of constructs harboring mutations at the 5' end of the U3 region in the 3' LTR was analyzed for particle export, reverse transcription, and replication. Following transient transfection some mutants were severely impaired in generating infectious virus, while others replicated almost like the wild type. The replication competence of the mutants was unrelated to the cleavability of the newly created U3 end. This became obvious with two mutants both belonging to the high-titer type. One mutant containing a dinucleotide artificially transferred from the right to the left end was trimmed upon integration, while another one with an unrelated dinucleotide in that place was not. The latter construct in particular showed that the canonical TG motif at the beginning of the provirus is not essential for foamy virus integration.

FIG. 1.

Amounts of cell-free virus produced by cells transfected with proviral plasmids and titer determination by the LacZ assay (39). The results from four independent experiments and standard deviations (error bars) are shown.

FIG. 2.

Vector constructs and transfer rates. 293T cells were transfected with the replication-deficient vector pMH123 or its derivatives with mutations of the 3′ LTR along with an Env-expressing plasmid. The transduction rates of HT1080 target cells expressed as mean percentages (± standard deviation) from four experiments indicated that the mutants transduced recipient cells less efficiently than wild-type virus-derived vector.

FIG. 3.

Overview of the results obtained upon sequencing the two-LTR junctions of mutant viruses. The figures on the left indicate the numbers of clones with unaltered sequences (first line), isolated U5-end deletions (second line), isolated U3 deletions (third line), deletions of both ends (forth line), and insertions regardless of additional deletions (fifth line) of the total number of clones analyzed. The two-LTR sequences are available as supplementary information at http://www.uni-wuerzburg.de/virologie/PFV2LTRsequences.html.