Generation of an improved foamy virus vector by dissection of cis-acting sequences

Abstract

In contrast to other retroviruses, foamy viruses (FVs) generate their Pol protein precursor independently of the Gag protein from a spliced mRNA. The exact mechanism of Pol protein incorporation into the viral capsid is poorly understood. Previously, we showed that Pol encapsidation critically depends on the packaging of (pre-) genomic RNA and identified two distinct signals within the cis-acting sequences (CASI and CASII), Pol encapsidation sequences (PESI and PESII), which are required for Pol capsid incorporation. Here, we investigated whether the presence of PESI and PESII in an FV vector is sufficient for Pol encapsidation and whether the rather extended CASII element can be shortened without loss of functionality. Our results indicate that (i) the presence of PESI and II are not sufficient for Pol encapsidation, (ii) prototype FV vectors with a shortened CASII element retain Pol incorporation and full functionality, in particular upon transducing fibroblasts and primary human mesenchymal stem cells, (iii) the presence of the central poly purine tract significantly increased the transduction rates of FV vectors and (iv) Pol encapsidation and RNA packaging can be clearly separated. In essence, we designed a new FV vector that bears approximately 850 bp less of CAS than previously established vectors and is fully functional when analysed to transduce cell lines and primary human cells. INTRODUCTIONSpumaretroviruses or foamy viruses (FVs) constitute one of the two subfamilies of retroviruses (Linial et al., 2005). The replication pathway of FV differs from what has been established for orthoretroviruses (Linial, 2007;Rethwilm, 2005). Despite the conservation of the general physical order of the provirus with long-terminal repeat (LTR)-gag-pol-env-accessory genes-LTR virtually all aspects of the replication pathway have their own facets that separate FVs from orthoretroviruses (Rethwilm, 2003). Among these, the ways that FVs express and encapsidate their reverse transcriptase proteins are central and pose some unresolved questions. The FV pol gene is expressed independently of the gag gene essentially from a spliced mRNA and gives rise to a large precursor protein comprised of protease (PR), reverse transcriptase/RNaseH (RT) and integrase (IN) (Bodem et al., 1996;Enssle et al., 1996;Yu et al., 1996). While IN is cleaved from the rest of the protein, no cleavage of PR from RT occurs (Flügel & Pfrepper, 2003). The capsid protein-independent translation of the pol gene raises the question of how the Pol protein is incorporated into the newly formed capsid.FV genomes bear two cis-acting sequences (CASI and II), which are required for transfer of a vector (Erlwein et al., 1998;Heinkelein et al., 1998;Wu et al., 1998). These elements are located in the 59 region of the (pre-) genomic RNA extending some hundred nucleotides into the gag gene and in the 39 pol genomic region (Heinkelein et al., , 2002aRussell et al., 2001;Trobridge et al., 2002). Both regions (together more than 2.5 kb) have complex functions in virus ...

Rescued Chondrogenesis of Mesenchymal Stem Cells under Interleukin 1 Challenge by Foamyviral Interleukin 1 Receptor Antagonist Gene Transfer

“…All used foamy vector (FV) plasmids were derived from the plasmid MD9 (Heinkelein et al, 2002; Peters et al, 2005; Wiktorowicz et al, 2009) that expresses the enhanced green fluorescent protein (EGFP) markergene driven by the spleen focus forming virus (SFFV)-U3 promoter ( Figure 1A ). The FV plasmids NA1 and KG84 ( Figures 1A , 2A ) were already described earlier (Gartner et al, 2009; Armbruster et al, 2014).…”

Molecular biology of foamy viruses

“…Further investigations to extend the repertoire of diseases tackled by FV vectors were also performed [102][103][104].…”

Progress and prospects: Foamy virus vectors enter a new age

“…With no packaging cell line available, strategies to produce foamy virus vectors depend on transient co-transfection of up to three individual expression cassettes for the structural proteins Gag, Pol and Env and a plasmid encoding the transgene. 25 This minimizes the generation of replication-competent PFV through recombination, improving safety. Replication-defective foamy virus vectors can now be produced consistently at titres of 10 7 ml after concentration, sufficient for ex vivo gene therapy applications, and without detectable helper virus.…”