DNA strand breaks and gaps target retroviral intasome binding and integration.

Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration. Using single molecule Förster resonance energy transfer (smFRET), we show prototype foamy virus (PFV) intasomes specifically bind to DNA strand breaks and gaps. These break and gap DNA discontinuities mimic oxidative base excision repair (BER) lesion-processing intermediates that have been shown to affect retrovirus integration in vivo. The increased DNA binding events targeted strand transfer to the break/gap site without inducing substantial intasome conformational changes. The major oxidative BER substrate 8-oxo-guanine as well as a G/T mismatch or +T nucleotide insertion that typically introduce a bend or localized flexibility into the DNA, did not increase intasome binding or targeted integration. These results identify DNA breaks or gaps as modulators of dynamic intasome-target DNA interactions that encourage site-directed integration.

PREB inhibits the replication of prototype foamy virus by affecting its transcription.

BACKGROUND: Foamy viruses (FVs) are unique nonpathogenic retroviruses, which remain latent in the host for a long time. Therefore, they may be safe, effective gene transfer vectors. In this study, were assessed FV-host cell interactions and the molecular mechanisms underlying FV latent infection. METHODS: We used the prototype FV (PFV) to infect HT1080 cells and a PFV indicator cell line (PFVL) to measure virus titers. After 48 h of infection, the culture supernatant (i.e., cell-free PFV particles) and transfected cells (i.e., cell-associated PFV particles) were harvested and incubated with PFVL. After another 48 h, the luciferase activity was used to measure virus titers. RESULTS: Through transcriptomics sequencing, we found that PREB mRNA expression was significantly upregulated. Moreover, PREB overexpression reduced PFV replication, whereas endogenous PREB knockdown increased PFV replication. PREB interacted with the Tas DNA-binding and transcriptional activation domains and interfered with its binding to the PFV long terminal repeat and internal promoter, preventing the recruitment of transcription factors and thereby inhibiting the transactivation function of Tas. PREB C-terminal 329-418 aa played a major role in inhibiting PFV replication; PREB also inhibited bovine FV replication. Therefore, PREB has a broad-spectrum inhibitory effect on FV replication. CONCLUSIONS: Our results demonstrated that PREB inhibits PFV replication by impeding its transcription.

Effects of Chemokine Ligand 2 on Budding of Bovine Foamy Virus.

The endosomal sorting complex required for transport (ESCRT) machinery is essential for the budding of retroviruses such as human immunodeficiency virus (HIV) and bovine foamy virus (BFV), which rely on their late domain to recruit ESCRT complexes to facilitate budding. However, the impact of intracellular host proteins on BFV budding remains poorly understood. In this study, we aimed to investigate the impact of CCL2 on BFV budding and interactions with key host proteins. Our results indicate that CCL2 promotes BFV budding in an ALG-2-interacting protein X (Alix)-dependent manner by enhancing the interaction between Alix and BFV Gag (BGag). Notably, we found a link between Alix, BGag and CCL2, with Alix mediating the interaction between the latter two. Furthermore, we observed that natural host bovine CCL2 also has a facilitating role in the budding process of BFV, similar to human CCL2. Taken together, these results demonstrate that CCL2 promotes BFV budding by enhancing the Alix-BGag association.

SGK1, a Serine/Threonine Kinase, Inhibits Prototype Foamy Virus Replication.

Foamy viruses (FVs) are complex retroviruses belonging to the Spumaretrovirinae subfamily of the Retroviridae family. In contrast to human immunodeficiency virus (HIV), another member of the Retroviridae family, FVs are nonpathogenic in their natural hosts or in experimentally infected animals. Prototype foamy virus (PFV) is the only foamy virus that can infect humans through cross-species transmission and does not show any pathogenicity after infection. Consequently, PFV is considered a safe and efficient gene transfer vector. Understanding the host proteins involved in the replication of PFV and the mechanism of interaction between the host and the virus might lead to studies to improve the efficiency of gene transfer. To date, only a few host factors have been identified that affect PFV replication. In the present study, we report that PFV infection enhances the promoter activity of SGK1 (encoding serum/glucocorticoid regulated kinase 1) via the Tas protein signaling pathway, and then upregulates the mRNA and protein levels of SGK1. Overexpression of SGK1 reduced PFV replication, whereas its depletion using small interfering RNA increased PFV replication. SGK1 inhibits PFV replication by impairing the function of the PFV Tas activation domain in a kinase-independent manner and reducing the stability of the Gag protein in a kinase-dependent manner. In addition, both human and bovine SGK1 proteins inhibit the replication of bovine foamy virus (BFV) and PFV. These findings not only improved our understanding of the function of SGK1 and its relationship with foamy viruses, but also contributed to determining the antiviral mechanism of the host. IMPORTANCE Foamy viruses can integrate into the host chromosome and are nonpathogenic in natural hosts or in experimentally infected animals. Therefore, foamy viruses are considered to be safe and efficient gene transfer vectors. Persistent infection of foamy viruses is partly caused by the restrictive effect of host factors on the virus. However, only a few cellular proteins are known to influence the replication of foamy viruses. In this study, we report that SGK1 inhibits the replication of prototype foamy virus by affecting the function of the transcription activator, Tas, and reducing the stability of the structural protein, Gag. These results will increase our understanding of the interaction between the virus and host factors, deepening our perception of host antiviral defenses and the function of SGK1, and could improve the gene transfer efficiency of foamy viruses.

Characterization of Bovine Foamy Virus Gag Late Assembly Domain Motifs and Their Role in Recruiting ESCRT for Budding.

A large number of retroviruses, such as human immunodeficiency virus (HIV) and prototype foamy virus (PFV), recruit the endosomal sorting complex required for transport (ESCRT) through the late domain (L domain) on the Gag structural protein for virus budding. However, little is known about the molecular mechanism of bovine foamy virus (BFV) budding. In the present study, we report that BFV recruits ESCRT for budding through the L domain of Gag. Specifically, knockdown of VPS4 (encoding vacuolar protein sorting 4), ALIX (encoding ALG-2-interacting protein X), and TSG101 (encoding tumor susceptibility 101) indicated that BFV uses ESCRT for budding. Mutational analysis of BFV Gag (BGag) showed that, in contrast to the classical L domain motifs, BGag contains two motifs, P56LPI and Y103GPL, with L domain functions. In addition, the two L domains are necessary for the cytoplasmic localization of BGag, which is important for effective budding. Furthermore, we demonstrated that the functional site of Alix is V498 in the V domain and the functional site of Tsg101 is N69 in the UBC-like domain for BFV budding. Taken together, these results demonstrate that BFV recruits ESCRT for budding through the PLPI and YGPL L domain motifs in BGag.

Inhibition of cell proliferation by Tas of foamy viruses through cell cycle arrest or apoptosis underlines the different mechanisms of virus-host interactions.

Foamy viruses belong to the Spumaretrovirinae subfamily member of the Retroviridae family and produce nonpathogenic infection to hosts in the natural conditions. However, infections of foamy viruses can dramatically cause severe cytopathic effects in vitro. To date, the exact molecular mechanism has remained unclear which implied the tremendous importance of virus-host cell immune reactions. In this study, we found that the transactivator Tas in two foamy viruses isolated from Old World Monkey (OWM) induced obvious inhibition of cell proliferation via the upregulation of Foxo3a expression. It was mediated by the generation of ROS and the initiation of ER stress, and ultimately, the mitochondrial apoptosis pathway was triggered. Notably, PFV Tas contributed to the accumulation of G0/G1 phase cycle arrest induced by the activation of the p53 signaling pathway and the nuclear transportation of HDAC4 via upregulating PPM1E expression. Together, these results demonstrated the different survival strategies by which foamy virus can hijack host cell cytokines and regulate virus-host cell interactions.

Coelacanth SERINC2 Inhibits HIV-1 Infectivity and Is Counteracted by Envelope Glycoprotein from Foamy Virus.

SERINC5 restricts nef-defective HIV-1 by affecting early steps of the virus life cycle. Distantly related retroviruses with a wide host range encode virulent factors in response to challenge by SERINC5. However, the evolutionary origins of this antiretroviral activity, its prevalence among the paralogs, and its ability to target retroviruses remain understudied. In agreement with previous studies, we found that four human SERINC paralogs inhibit nef-defective HIV-1, with SERINC2 being an exception. Here, we demonstrate that this lack of activity in human SERINC2 is associated with its post-whole-genome duplication (post-WGD) divergence, as evidenced by the ability of pre-WGD orthologs from Saccharomyces cerevisiae and flies and a post-WGD-proximate SERINC2 from coelacanths to inhibit the virus. Intriguingly, Nef is unable to counter coelacanth SERINC2, indicating that such activity was directed toward other retroviruses found in coelacanths (like foamy viruses). However, foamy virus-derived vectors are intrinsically resistant to the action of SERINC2, and we show that the foamy virus envelope confers this resistance by affecting its steady-state levels. Our study highlights an ancient origin of antiretroviral activity in SERINCs and a hitherto-unknown interaction with a foamy virus. IMPORTANCESERINC5 constitutes a critical barrier to the propagation of retroviruses, as highlighted by parallel emergence of anti-SERINC5 activities among distant retroviral lineages. Therefore, understanding the origin and evolution of these host factors will provide key information about virus-host relationships that can be exploited for future drug development. Here, we show that SERINC5-mediated nef-defective HIV-1 infection inhibition is evolutionarily conserved. SERINC2 from coelacanth restricts HIV-1, and it was functionally adapted to target foamy viruses. Our findings provide insights into the evolutionary origin of antiretroviral activity in the SERINC gene family and uncover the role of SERINCs in shaping the long-term conflicts between retroviruses and their hosts.

Retroviral prototype foamy virus intasome binding to a nucleosome target does not determine integration efficiency.

Retroviral integrases must navigate host DNA packaged as chromatin during integration of the viral genome. Prototype foamy virus (PFV) integrase (IN) forms a tetramer bound to two viral DNA (vDNA) ends in a complex termed an intasome. PFV IN consists of four domains: the amino terminal extension domain (NED), amino terminal domain (NTD), catalytic core domain (CCD), and carboxyl terminal domain (CTD). The domains of the two inner IN protomers have been visualized, as well as the CCDs of the two outer IN protomers. However, the roles of the amino and carboxyl terminal domains of the PFV intasome outer subunits during integration to a nucleosome target substrate are not clear. We used the well-characterized 601 nucleosome to assay integration activity as well as intasome binding. PFV intasome integration to 601 nucleosomes occurs in clusters at four independent sites. We find that the outer protomer NED and NTD domains have no significant effects on integration efficiency, site selection, or binding. The CTDs of the outer PFV intasome subunits dramatically affect nucleosome binding but have little effect on total integration efficiency. The outer PFV IN CTDs did significantly alter the integration efficiency at one site. Histone tails also significantly affect intasome binding, but have little impact on PFV integration efficiency or site selection. These results indicate that binding to nucleosomes does not correlate with integration efficiency and suggests most intasome-binding events are unproductive.

HIV-2 Vif and foamy virus Bet antagonize APOBEC3B by different mechanisms.

The family of human APOBEC3 (A3) restriction factors is formed by seven different proteins, A3A-D and A3F-H. Among these A3s, A3B harbors strong restriction activity against several retroviruses, such as SIV, and MLV. How lentiviruses and other retroviruses, prevalent in many primate species, counteract A3B is poorly understood. In this study, we found that A3B strongly inhibited SIVmac and HIV-2 infectivity, which was antagonized by their Vif proteins. Both SIVmac and HIV-2 Vifs diminished the protein level of A3B in viral producer cells, and hindered A3B incorporation into viral particles. We observed that HIV-2 Vif binds A3B and induces its degradation by assembly of an A3-Vif-CUL5-ElonginB/C E3-ligase complex. A3B and HIV-2 Vif localize and interact in the nucleus. In addition, we also found that the accessory protein Bet of prototype foamy virus (PFV) significantly antagonized the anti-SIVmac activity of A3B. Like Vif, Bet prevented the incorporation of A3B into viral particles. However, in contrast to Vif Bet did not induce the degradation of A3B. Rather, Bet binds A3B to block formation of high molecular weight A3B complexes and induces A3B cytoplasmic trapping. In summary, these findings indicate that A3B is recognized by diverse retroviruses and counteracted by virus-specific pathways that could be targeted to inhibit A3B mutating activity in cancers.

Prototype foamy virus downregulates RelB expression to facilitate viral replication.

Foamy viruses (FVs) are classified in the subfamily Spumaretrovirinae and bridge the gap between Orthoretrovirinae and Hepadnaviridae. FVs have strong cytopathic effects against cells cultured in vitro. However, they establish lifelong latent infections without evident pathology in the host. The roles of cellular factors in FV replication are poorly understood. To better understand this area, we determined the transcriptomes of HT1080 cells infected with prototype foamy virus (PFV) to measure the effect of PFV infection on the expression of cellular genes. We found that the level of RelB mRNA, a member of the nuclear factor-κB (NF-κB) protein family, was significantly decreased as a result of PFV infection, and this was further confirmed with real-time PCR. Interestingly, overexpression of RelB reduced PFV replication, whereas its depletion using small interfering RNA increased PFV replication. This inhibitory effect of RelB results from diminished transactivation of the viral long terminal repeat (LTR) promoter and an internal promoter (IP) by viral Tas protein. Together, these data demonstrate that PFV infection downregulates the viral inhibitory host factor RelB, which otherwise restricts viral gene expression.

The Late Domain of Prototype Foamy Virus Gag Facilitates Autophagic Clearance of Stress Granules by Promoting Amphisome Formation.

Prototype foamy virus (PFV), a complex retrovirus belonging to Spumaretrovirinae, maintains lifelong latent infection. The maintenance of lifelong latent infection by viruses relies on the repression of the type I interferon (IFN) response. However, the mechanism involving PFV latency, especially regarding the suppression of the IFN response, is poorly understood. Our previous study showed that PFV promotes autophagic flux. However, the underlying mechanism and the role of PFV-induced autophagy in latent infection have not been clarified. Here, we report that the PFV viral structural protein Gag induced amphisome formation and triggered autophagic clearance of stress granules (SGs) to attenuate type I IFN production. Moreover, the late domain (L-domain) of Gag played a central role in Alix recruitment, which promoted endosomal sorting complex required for transport I (ESCRT-I) formation and amphisome accumulation by facilitating late endosome formation. Our data suggest that PFV Gag represses the host IFN response through autophagic clearance of SGs by activating the endosome-autophagy pathway. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.IMPORTANCE Maintenance of lifelong latent infection for viruses relies on repression of the type I IFN response. Autophagy plays a double-edged sword in antiviral immunity. However, the role of autophagy in the regulation of the type I IFN response and the mechanism involving virus-promoted autophagy have not been fully elucidated. SGs are an immune complex associated with the antiviral immune response and are critical for type I IFN production. Autophagic clearance of SGs is one means of degradation of SGs and is associated with regulation of immunity, but the detailed mechanism remains unclear. In this article, we demonstrate that PFV Gag recruits ESCRT-I to facilitate amphisome formation. Our data also suggest that amphisome formation is a critical event for autophagic clearance of SGs and repression of the type I IFN response. More importantly, we found a novel mechanism by which a retrovirus inhibits the SG response to repress the type I IFN response.

Structural Insights on Retroviral DNA Integration: Learning from Foamy Viruses.

Foamy viruses (FV) are retroviruses belonging to the Spumaretrovirinae subfamily. They are non-pathogenic viruses endemic in several mammalian hosts like non-human primates, felines, bovines, and equines. Retroviral DNA integration is a mandatory step and constitutes a prime target for antiretroviral therapy. This activity, conserved among retroviruses and long terminal repeat (LTR) retrotransposons, involves a viral nucleoprotein complex called intasome. In the last decade, a plethora of structural insights on retroviral DNA integration arose from the study of FV. Here, we review the biochemistry and the structural features of the FV integration apparatus and will also discuss the mechanism of action of strand transfer inhibitors.

Nucleosome DNA unwrapping does not affect prototype foamy virus integration efficiency or site selection.

Eukaryotic DNA binding proteins must access genomic DNA that is packaged into chromatin in vivo. During a productive infection, retroviral integrases (IN) must similarly interact with chromatin to integrate the viral cDNA genome. Here we examine the role of nucleosome DNA unwrapping in the retroviral integrase search for a target site. These studies utilized PFV intasomes that are comprised of a tetramer of PFV IN with two oligomers mimicking the viral cDNA ends. Modified recombinant human histones were used to generate nucleosomes with increased unwrapping rates at different DNA regions. These modifications included the acetylmimetic H3(K56Q) and the chemically engineered H4(K77ac, K79ac). While transcription factors and DNA damage sensors may search nucleosome bound DNA during transient unwrapping, PFV intasome mediated integration appears to be unaffected by increased nucleosome unwrapping. These studies suggest PFV intasomes do not utilize nucleosome unwrapping to search nucleosome targets.

Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.

Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells.

Important role of N108 residue in binding of bovine foamy virus transactivator Tas to viral promoters.

BACKGROUND: Bovine foamy virus (BFV) encodes the transactivator BTas, which enhances viral gene transcription by binding to the long terminal repeat promoter and the internal promoter. In this study, we investigated the different replication capacities of two similar BFV full-length DNA clones, pBS-BFV-Y and pBS-BFV-B. RESULTS: Here, functional analysis of several chimeric clones revealed a major role for the C-terminal region of the viral genome in causing this difference. Furthermore, BTas-B, which is located in this C-terminal region, exhibited a 20-fold higher transactivation activity than BTas-Y. Sequence alignment showed that these two sequences differ only at amino acid 108, with BTas-B containing N108 and BTas-Y containing D108 at this position. Results of mutagenesis studies demonstrated that residue N108 is important for BTas binding to viral promoters. In addition, the N108D mutation in pBS-BFV-B reduced the viral replication capacity by about 1.5-fold. CONCLUSIONS: Our results suggest that residue N108 is important for BTas binding to BFV promoters and has a major role in BFV replication. These findings not only advances our understanding of the transactivation mechanism of BTas, but they also highlight the importance of certain sequence polymorphisms in modulating the replication capacity of isolated BFV clones.

Nuclear import of prototype foamy virus transactivator Bel1 is mediated by KPNA1, KPNA6 and KPNA7.

Bel1, a transactivator of the prototype foamy virus (PFV), plays pivotal roles in the replication of PFV. Previous studies have demonstrated that Bel1 bears a nuclear localization signal (NLS); however, its amino acid sequence remains unclear and the corresponding adaptor importins have not yet been identified. In this study, we inserted various fragments of Bel1 into an EGFP-GST fusion protein and investigated their subcellular localization by fluorescence microscopy. We found that the 215PRQKRPR221 fragment, which accords with the consensus sequence K(K/R)X(K/R) of the monopartite NLS, directed the nuclear translocation of Bel1. Point mutation experiments revealed that K218, R219 and R221 were essential for the nuclear localization of Bel1. The results of GST pull-down assay revealed that the Bel1 peptide 215-221, which bears the NLS, interacted with the nucleocytoplasmic transport receptors, karyopherin alpha 1 (importin alpha 5) (KPNA1), karyopherin alpha 6 (importin alpha 7) (KPNA6) and karyopherin alpha 7 (importin alpha 8) (KPNA7). Finally, in vitro nuclear import assays demonstrated that KPNA1, KPNA6 or KPNA7, along with other necessary nuclear factors, caused Bel1 to localize to the nucleus. Thus, the findings of our study indicate that KPNA1, KPNA6 and KPNA7 are involved in Bel1 nuclear distribution.

Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.

Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3'-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3'-hydroxyls into the target DNA separated by 4-6 bp. Host DNA repair restores the resulting 5'-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration.

Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication.

Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle.

Structural basis for retroviral integration into nucleosomes.

Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.

Efficient transient genetic manipulation in vitro and in vivo by prototype foamy virus-mediated nonviral RNA transfer.

Vector systems based on different retroviruses are widely used to achieve stable integration and expression of transgenes. More recently, transient genetic manipulation systems were developed that are based on integration- or reverse transcription-deficient retroviruses. Lack of viral genome integration is desirable not only for reducing tumorigenic potential but also for applications requiring transient transgene expression such as reprogramming or genome editing. However, all existing transient retroviral vector systems rely on virus-encoded encapsidation sequences for the transfer of heterologous genetic material. We discovered that the transient transgene expression observed in target cells transduced by reverse transcriptase-deficient foamy virus (FV) vectors is the consequence of subgenomic RNA encapsidation into FV particles. Based on this initial observation, we describe here the establishment of FV vectors that enable the efficient transient expression of various transgenes by packaging, transfer, and de novo translation of nonviral RNAs both in vitro and in vivo. Transient transgene expression levels were comparable to integrase-deficient vectors but, unlike the latter, declined to background levels within a few days. Our results show that this new FV vector system provides a useful, novel tool for efficient transient genetic manipulation of target tissues by transfer of nonviral RNAs.