Efficient intracellular retrotransposition of an exogenous primate retrovirus genome

Abstract

The foamy virus (FV) subgroup of Retroviridae reverse transcribe their RNA (pre-)genome late in the replication cycle before leaving an infected cell. We studied whether a marker gene-transducing FV vector is able to shuttle to the nucleus and integrate into host cell genomic DNA. While a potential intracellular retrotransposition of vectors derived from other retroviruses was below the detection limit of our assay, we found that up to 5% of cells transfected with the FV vector were stably transduced, harboring 1 to~10 vector integrants. Generation of the integrants depended on expression of functional capsid, reverse transcriptase and integrase proteins, and did not involve an extracellular step. PCR analysis of the U3 region of the 5¢ long terminal repeat and determination of proviral integration sites showed that a reverse transcription step had taken place to generate the integrants. Co-expression of a mutated envelope allowing particle egress and avoiding extracellular infection resulted in a signi®cantly increased rescue of cells harboring integrants, suggesting that accumulation of proviruses via intracellular retrotransposition represents an integral part of the FV replication strategy.

Foamy virus–adenovirus hybrid vectors

“…11 The efficiency of PFV IRT was found to be in the percentage range, which is far above any mammalian retrotransposon. 11 We therefore wished to characterize hybrids between the nonintegrating high-capacity Adand PFV-derived vectors to investigate the possibility of establishing prototype hybrid vectors that combine the features of a high titer production and persistence. To this end, we attempted to exploit the extracellular as well as the intracellular replication pathway of PFV.…”

“…PFV-specific IRT has been shown to consist of the conversion of an RNA pregenome into an integration-competent cDNA copy without the generation of an infectious extracellular virus. 11 PFV-specific IRT depends on functionally active Gag and Pol proteins. 11 The efficiency of PFV IRT was found to be in the percentage range, which is far above any mammalian retrotransposon.…”

“…11 PFV-specific IRT depends on functionally active Gag and Pol proteins. 11 The efficiency of PFV IRT was found to be in the percentage range, which is far above any mammalian retrotransposon. 11 We therefore wished to characterize hybrids between the nonintegrating high-capacity Adand PFV-derived vectors to investigate the possibility of establishing prototype hybrid vectors that combine the features of a high titer production and persistence.…”

“…16,17 The PFV part of pFAD-7 can only perform an intracellular replication cycle, that is, retrotranspose the PFV genome into that of the host cell. Since we expected the efficiency of stable marker gene transduction by pFAD-7 to be far less compared with pFAD-2, 11 we also generated a negative control, pFAD-8 ( Figure 1), in which the translational start codon of the pol gene was mutated from ATG to CTG. In contrast to ORVs, in spumaretroviruses, the Pol protein is not expressed as a fusion product with the Gag capsid protein, but independently of a spliced pol RNA.…”

RNAi and retroviruses: are they in RISC?

“…Is required PFV-1 (81) HIV (81) (96) LTR and non-LTR retroelements (97) Has no effect on HIV-1, SIVmac, MLV, and M-PMV (97) and this retrotransposition depends on the expression of a functional GAG protein (109) . As AGO2 affects PFV-1 replication (81) , presumably at or before the GAG multimerization step (unpublished data), it may also influence PFV-1 retrotransposition.…”